RESUMO
Peiminine, the primary biologically active compound from Fritillaria thunbergii Miq., has demonstrated significant pharmacological activities. Doxorubicin is one of the most potent chemotherapeutic agents for breast cancer (BC). This study was designed to investigate the efficacy and underlying mechanisms of Peiminine combined with Doxorubicin in treating BC. Our results demonstrated that the combination of Peiminine and 1â¯mg/kg Doxorubicin exhibited more significant suppression of tumor growth compared with the monotherapy in MDA-MB-231 xenograft nude mice model, which is comparable to the effect of 3â¯mg/kg Doxorubicin in vivo. Notably, the 3â¯mg/kg Doxorubicin monotherapy resulted in organ toxicity, specifically in the liver and heart, whereas no toxicity was observed in the combination group. In vitro, this combined treatment exhibited a synergistic reduction on the viability of BC cells. Peiminine enhanced the cell cycle arrest and DNA damage induced by Doxorubicin. Furthermore, the combination treatment effectively blocked DNA repair by inhibiting the MAPKs signaling pathways. And ZEB1 knockdown attenuated the combined effect of Peiminine and Doxorubicin on cell viability and DNA damage. In conclusion, our study found that the combination of Peiminine and Doxorubicin showed synergistic inhibitory effects on BC both in vivo and in vitro through enhancing Doxorubicin-induced DNA damage. These findings support that their combination is a novel and promising therapeutic strategy for treating BC.
Assuntos
Neoplasias da Mama , Cevanas , Camundongos , Animais , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Camundongos Nus , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Adutos de DNA/farmacologia , Adutos de DNA/uso terapêutico , Linhagem Celular Tumoral , Apoptose , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
BACKGROUND: Oxaliplatin (Oxa) is the first-line chemotherapy drug for colorectal cancer (CRC), and Oxa resistance is crucial for treatment failure. Prostaglandin F2α synthase (PGF2α) (PGFS), an enzyme that catalyzes the production of PGF2α, is involved in the proliferation and growth of a variety of tumors. However, the role of PGFS in Oxa resistance in CRC remains unclear. AIM: To explore the role and related mechanisms of PGFS in mediating Oxa resistance in CRC. METHODS: The PGFS expression level was examined in 37 pairs of CRC tissues and paracancerous tissues at both the mRNA and protein levels. Overexpression or knockdown of PGFS was performed in CRC cell lines with acquired Oxa resistance (HCT116-OxR and HCT8-OxR) and their parental cell lines (HCT116 and HCT8) to assess its influence on cell proliferation, chemoresistance, apoptosis, and DNA damage. For determination of the underlying mechanisms, CRC cells were examined for platinum-DNA adducts and reactive oxygen species (ROS) levels in the presence of a PGFS inhibitor or its products. RESULTS: Both the protein and mRNA levels of PGFS were increased in the 37 examined CRC tissues compared to the adjacent normal tissues. Oxa induced PGFS expression in the parental HCT116 and HCT8 cells in a dose-dependent manner. Furthermore, overexpression of PGFS in parental CRC cells significantly attenuated Oxa-induced proliferative suppression, apoptosis, and DNA damage. In contrast, knockdown of PGFS in Oxa-resistant HCT116 and HCT8 cells (HCT116-OxR and HCT8-OxR) accentuated the effect of Oxa treatment in vitro and in vivo. The addition of the PGFS inhibitor indomethacin enhanced the cytotoxicity caused by Oxa. Treatment with the PGFS-catalyzed product PGF2α reversed the effect of PGFS knockdown on Oxa sensitivity. Interestingly, PGFS inhibited the formation of platinum-DNA adducts in a PGF2α-independent manner. PGF2α exerts its protective effect against DNA damage by reducing ROS levels. CONCLUSION: PGFS promotes resistance to Oxa in CRC via both PGF2α-dependent and PGF2α-independent mechanisms.
Assuntos
Neoplasias Colorretais , Platina , Humanos , Oxaliplatina/farmacologia , Oxaliplatina/uso terapêutico , Platina/farmacologia , Platina/uso terapêutico , Adutos de DNA/farmacologia , Adutos de DNA/uso terapêutico , Espécies Reativas de Oxigênio , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , RNA Mensageiro/metabolismo , Prostaglandinas , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular TumoralRESUMO
To further explore the pharmacological effect of pachymaran, this article studied the inhibition of pachymaran on oxidative stress and genetic damage induced by formaldehyde. 40 adult Kunming male mice were randomly divided into four groups with different interventions. One week later, the contents of serum SOD, GR, MDA, DNA-protein crosslink (DPC), 8-hydroxydeoxyguanosine (8-OHDG) and DNA adduct were determined by ELISA. The results showed that there were statistically significant differences in the contents of SOD, GR and MDA among the four groups (P < 0.01). The activity of SOD and GR increased along with the increase of pachymaran dosage (SOD: rs = 0.912, P < 0.01; GR: rs = 0.857, P < 0.01), while the content of MDA showing a significant negative correlation (rs = - 0.893, P < 0.01). There were statistically significant differences in the levels of DPC, 8-OHDG and DNA adduct among the four groups (DPC and DNA adduct: P < 0.01, 8-OHDG: P < 0.05), the concentration decreased along with the increase of pachymaran dosage (DPC: rs = - 0.855, P < 0.01; 8-OHDG:rs = - 0.412, P < 0.05, DNA adduct: γs = - 0.869, P < 0.01). It can be inferred that pachymaran can inhibit oxidative stress and DNA damage induced by formaldehyde with the dose-effect relationship.
Assuntos
Adutos de DNA , Dano ao DNA , Camundongos , Animais , Masculino , Adutos de DNA/farmacologia , 8-Hidroxi-2'-Desoxiguanosina , Estresse Oxidativo , Formaldeído/toxicidade , Proteínas/farmacologia , Superóxido Dismutase/metabolismo , DesoxiguanosinaRESUMO
Aflatoxin B1 (AFB1) is known to derange the hepatorenal system by redox, DNA adduct formation and apoptotic networks. Endogenous 3-indole propionic acid (3-IPA) is a metabolite of tryptophan metabolism by gut microbiota that can protect against redox imbalance, inflammation and cellular lipid damage. We investigated the beneficial effect of 3-IPA against AFB1-mediated organ toxicity in male rats post 28 days of consecutive treatment. The 3-IPA (25 and 50 mg/kg) was orally administered alongside AFB1 (50 µg/kg) treatment. Biochemical and enzyme-linked immunosorbent assays were utilised to examine biomarkers of hepatorenal function, oxidative status and inflammation. DNA damage and apoptosis were also assessed, and histological staining techniques were used to investigate hepatorenal tissues for pathological indicators. The 3-IPA supplementation abated AFB1-mediated increases in biomarkers of hepatic and renal dysfunction in rat serum. Co-administration of 3-IPA further reduced AFB1-induced redox imbalance (by upregulating antioxidant mediators and enzymes [GSH, TSH, Trx, Trx-R, SOD, CAT, GPx and GST]; reducing reactive oxygen species, lipid peroxidation and DNA adduct [RONS, LPO and 8-OH-dG] formation; suppressing pro-inflammatory and apoptotic mediators [XO, MPO, NO, IL-1ß and Casp -9 and -3]; and upregulating the level of interleukin 10 (IL-10). Moreover, treatment with 3-IPA lessened hepatorenal tissue injuries. These findings suggest that augmenting 3-IPA endogenously from tryptophan metabolism may provide a novel strategy to forestall xenobiotics-mediated hepatorenal toxicity, including AFB1.
Assuntos
Aflatoxina B1 , Adutos de DNA , Ratos , Animais , Masculino , Aflatoxina B1/toxicidade , Aflatoxina B1/metabolismo , Adutos de DNA/metabolismo , Adutos de DNA/farmacologia , Triptofano/metabolismo , Triptofano/farmacologia , Glutationa/metabolismo , Fígado , Inflamação/metabolismo , Rim/metabolismo , Biomarcadores/metabolismo , Estresse OxidativoRESUMO
The fungal toxin aflatoxin B1 (AB1) and its reactive intermediate, aflatoxin B1-8, 9 epoxide, could cause liver cancer by inducing DNA adducts. AB1 exposure can induce changes in the expression of several cancer-related genes. In this study, the effect of AB1 exposure on breast cancer MCF7 and normal breast MCF10A cell lines at the phenotypic and epigenetic levels was investigated to evaluate its potential in increasing the risk of breast cancer development. We hypothesized that, even at low concentrations, AB1 can cause changes in the expression of important genes involved in four pathways, i.e., p53, cancer, cell cycle, and apoptosis. The transcriptomic levels of BRCA1, BRCA2, p53, HER1, HER2, cMyc, BCL2, MCL1, CCND1, WNT3A, MAPK1, MAPK3, DAPK1, Casp8, and Casp9 were determined in MCF7 and MCF10A cells. Our results illustrate that treating both cells with AB1 induced cytotoxicity and apoptosis with reduction in cell viability in a concentration-dependent manner. Additionally, AB1 reduced reactive oxygen species levels. Phenotypically, AB1 caused cell-cycle arrest at G1, hypertrophy, and increased cell migration rates. There were changes in the expression levels of several tumor-related genes, which are known to contribute to activating cancer pathways. The effects of AB1 on the phenotype and epigenetics of both MCF7 and MCF10A cells associated with cancer development observed in this study suggest that AB1 is a potential risk factor for developing breast cancer.
Assuntos
Aflatoxina B1 , Proteína Supressora de Tumor p53 , Aflatoxina B1/toxicidade , Apoptose/genética , Linhagem Celular Tumoral , Adutos de DNA/farmacologia , Compostos de Epóxi/farmacologia , Humanos , Células MCF-7 , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Fenótipo , Espécies Reativas de Oxigênio/farmacologia , Proteína Supressora de Tumor p53/genéticaRESUMO
Pyrrolizidine alkaloids (PAs) have been found in over 6000 plants worldwide and represent the most common hepatotoxic phytotoxins. Catalyzed by hepatic cytochrome P450 enzymes, PAs are metabolized into reactive pyrrolic metabolites, which can alkylate cellular proteins and DNA to form pyrrole-protein adducts and pyrrole-DNA adducts, leading to cytotoxicity, genotoxicity, and tumorigenicity. To date, the correlation between these PA-derived pyrrole-protein and pyrrole-DNA adducts has not been well investigated. Retrorsine is a representative hepatotoxic and carcinogenic PA. In the present study, the correlations among the PA-derived liver DNA adducts, liver protein adducts, and serum protein adducts in retrorsine-treated mice under different dosage regimens were studied. The results showed positive correlations among these adducts, in which serum pyrrole-protein adducts were more accessible and present in higher abundance, and thus could be used as a suitable surrogate biomarker for pyrrole-DNA adducts to indicate the genetic or carcinogenic risk posed by retrorsine.
Assuntos
Adutos de DNA , Alcaloides de Pirrolizidina , Animais , Carcinógenos/metabolismo , DNA/metabolismo , Adutos de DNA/metabolismo , Adutos de DNA/farmacologia , Fígado , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas/metabolismo , Pirróis/toxicidade , Alcaloides de Pirrolizidina/toxicidadeRESUMO
Exposure to environmental and occupational contaminants leads to lung cancer. 3-Nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one, 3-NBA) is a potential carcinogen in ambient air or diesel particulate matter. Studies have revealed that short-term exposure to 3-NBA induces cell death, reactive oxygen species activation, and DNA adduct formation and damage. However, details of the mechanism by which chronic exposure to 3-NBA influences lung carcinogenesis remain largely unknown. In this study, human lung epithelial BEAS-2B cells were continuously exposed to 0-10-µM 3-NBA for 6 months. NanoString analysis was conducted to evaluate gene expression in the cells, revealing that 3-NBA-mediated transformation results in a distinct gene expression signature including carbon cancer metabolism, metastasis, and angiogenesis. Alterations in tumor-promoting genes such as EREG (epiregulin), SOX9, E-cadherin, TWIST, and IL-6 were involved in epithelial cell aggressiveness. Kaplan-Meier plotter analyses indicated that increased EREG and IL-6 expressions in early-stage lung cancer cells are correlated with poor survival. In vivo xenografts on 3-NBA-transformed cells exhibited prominent tumor formation and metastasis. EREG knockout cells exposed to 3-NBA for a short period exhibited high apoptosis and low colony formation. By contrast, overexpression of EREG in 3-NBA-transformed cells markedly activated the PI3K/AKT and MEK/ERK signaling pathways, resulting in tumorigenicity. Furthermore, elevated IL-6 and EREG expressions synergistically led to STAT3 signaling activation, resulting in clonogenic cell survival and migration. Taken together, chronic exposure of human lung epithelial cells to 3-NBA leads to malignant transformation, in which the EREG signaling pathway plays a pivotal mediating role. ⢠Short-term exposure of lung epithelial cells to 3-NBA can lead to ROS production and cell apoptosis. ⢠Long-term chronic exposure to 3-NBA upregulates the levels of tumor-promoting genes such as EREG and IL-6. ⢠Increased EREG expression in 3-NBA-transformed cells markedly contributes to tumorigenesis through PI3K/AKT and MEK/ERK activation and synergistically enhances the IL-6/STAT3 signaling pathway, which promotes tumorigenicity.
Assuntos
Adutos de DNA , Neoplasias Pulmonares , Benzo(a)Antracenos , Caderinas/metabolismo , Carbono/metabolismo , Carbono/farmacologia , Carcinogênese/metabolismo , Carcinógenos , Transformação Celular Neoplásica/metabolismo , Adutos de DNA/metabolismo , Adutos de DNA/farmacologia , Epirregulina/genética , Epirregulina/metabolismo , Epirregulina/farmacologia , Células Epiteliais/metabolismo , Humanos , Interleucina-6/metabolismo , Pulmão/metabolismo , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/farmacologia , Material Particulado/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Nucleotide excision repair (NER) and cell cycle checkpoints impact the ability of the anti-cancer drug cisplatin to inhibit cell proliferation and induce cell death. Genetic studies have shown that both NER and cell cycle progression are impacted by the circadian clock, which has emerged as a novel pharmacological target for the treatment of various disease states. In this study, cultured human cell lines were treated with combinations of cisplatin and the circadian clock modulating compounds KS15 and SR8278, which enhance circadian clock transcriptional output by inhibiting the activities of the cryptochrome and REV-ERB proteins, respectively. Treatment of cells with KS15 and SR8278 protected cells against the anti-proliferative effects of cisplatin and increased the expression of NER factor XPA and cell cycle regulators Wee1 and p21 at the mRNA and protein level. Correlated with these molecular changes, KS15 and SR8278 treatment resulted in fewer unrepaired cisplatin-DNA adducts in genomic DNA and a higher fraction of cells in the G1 phase of the cell cycle. Thus, the use of pharmacological agents targeting the circadian clock could be a novel approach to modulate the responses of normal and cancer cells to cisplatin chemotherapy regimens.
Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Criptocromos/antagonistas & inibidores , Adutos de DNA/efeitos dos fármacos , Adutos de DNA/farmacologia , Reparo do DNA/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Células A549 , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relógios Circadianos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células HaCaT , Humanos , Isoquinolinas/farmacologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiofenos/farmacologia , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
Studies that aim to characterise the susceptibility of the ecologically relevant and non-model fish polar cod (Boreogadus saida) to model carcinogens are required. Polar cod were exposed under laboratory conditions for six months to control, 0.03 µg BaP/g fish/week and 0.3 µg BaP/g fish/week dietary benzo(a)pyrene (BaP), a reference carcinogen. The concentrations of the 3-OH-BaP bile metabolite and transcriptional responses of genes involved in DNA adduct recognition (xpc), helicase activity (xpd), DNA repair (xpf, rad51) and tumour suppression (tp53) were assessed after 0, 1, 3 and 6 months of exposure, alongside body condition indexes (gonadosomatic index, hepatosomatic index and condition factor). Micronuclei and nuclear abnormalities in blood and spleen, and liver histopathological endpoints were assessed at the end of the experiment. Fish grew steadily over the whole experiment and no mortality was recorded. The concentrations of 3-OH-BaP increased significantly after 1 month of exposure to the highest BaP concentration and after 6 months of exposure to all BaP concentrations showing the biotransformation of the mother compound. Nevertheless, no significant induction of gene transcripts involved in DNA damage repair or tumour suppression were observed at the selected sampling times. These results together with the absence of chromosomal damage in blood and spleen cells, the subtle increase in nuclear abnormalities observed in spleen cells and the low occurrence of foci of cellular alteration suggested that the exposure was below the threshold of observable effects. Taken together, the results showed that polar cod was not susceptible to carcinogenesis using the BaP exposure regime employed herein.
Assuntos
Carcinógenos , Gadiformes , Animais , Benzo(a)pireno/toxicidade , Bile , Adutos de DNA/farmacologiaRESUMO
Stimuli-responsive DNA-based nanostructures have emerged as promising vehicles for intelligent drug delivery. In this study, i-motif DNA-conjugated gold nanostars (GNSs) were fabricated in a facile manner as stimuli-responsive drug delivery systems (denoted as A-GNS/DNA/DOX) for the treatment of cancer via combined chemo-photothermal therapy. The i-motif DNA is sensitive to the environmental pH and can switch from a single-stranded structure to a C-tetrad (i-motif) structure as the environmental pH decreases from neutral (â¼7.4) to acidic (<6.0). The loaded drug can then be released along with the conformational changes. To enhance cellular uptake and improve cancer cell selectivity, the aptamer AS1411, which recognizes nucleolins, was employed as a targeting moiety. The A-GNS/DNA/DOX nanocomposites were found to be highly capable of photothermal conversion and exhibited photostability under near-infrared (NIR) irradiation, and the pH and NIR irradiation effectively triggered the drug-release behaviors. In addition, the A-GNS/DNA/DOX nanocomposites exhibited good biocompatibility. The targeting recognition enabled the A-GNS/DNA/DOX to exhibit higher cellular uptake and therapeutic efficiency than the GNS/DNA/DOX. Notably, under NIR irradiation, a synergistic effect between chemotherapy and photothermal therapy can be achieved with the proposed delivery system, which exhibits much higher therapeutic efficiency both in monolayer cancer cells and tumor spheroids as compared with a single therapeutic method. This study highlights the potential of GNS/DNA nanoassemblies for intelligent anticancer drug delivery and combined cancer therapy.
Assuntos
Adutos de DNA/química , Adutos de DNA/farmacologia , DNA/química , Doxorrubicina/química , Doxorrubicina/farmacologia , Ouro/química , Nanopartículas Metálicas/química , Nanoestruturas/química , Neoplasias/tratamento farmacológico , Células 3T3 , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Materiais Biocompatíveis/química , Linhagem Celular , Linhagem Celular Tumoral , Terapia Combinada/métodos , Sistemas de Liberação de Medicamentos/métodos , Liberação Controlada de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Células MCF-7 , Camundongos , Nanocompostos/química , Fototerapia/métodosRESUMO
Tumor-targeted drug delivery systems with stimuli-response drug release have been increasingly used to improve the therapeutic efficacy of antitumor drugs. Here, we report a specific molecular recognition activation drug nanoplatform based on specially designed DNA sensor-capped doxorubicin (DOX)-loaded mesoporous silica nanoparticles (MSNs), designated as specific molecular recognition-activated nanoparticle (SMRAN). DNA sensors on the targeted nanoparticles can trigger DOX release through a conformational switch induced by MUC-1. This causes a significant difference in cell viability between breast cancer MCF-7 and normal breast Hs578bst cells (24.8% and 86.0%). In vivo experiments showed that the tumor volume was reduced 1.5-times in the SMRAN treatment group. Compared with that in the DOX group, due to significantly improved tumor accumulation and retention of DOX. The strategy of the MUC-1 activated drug delivery system is expected to provide a new perspective for clinical application.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Mucina-1/genética , Nanopartículas/química , Mama/efeitos dos fármacos , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Adutos de DNA/química , Adutos de DNA/farmacologia , Doxorrubicina/química , Doxorrubicina/farmacologia , Portadores de Fármacos , Liberação Controlada de Fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7RESUMO
Mitochondria-targeted nanoparticles, such as liposomes, polymers and inorganic particles, suffer from heterogeneity, low biocompatibility and low drug loading efficiency. Here, we present a novel delivery platform based on tetrahedral DNA nanostructures (TDNs) that enable the mitochondrial transportation of the anticancer drug doxorubicin (DOX) for cancer therapy. In our design, DOX was intercalated into TDNs, which executed the cell-killing function inside the tumor cells. Various numbers of d-(KLAKLAK)2 (KLA) were conjugated to TDNs to achieve the mitochondria targeting effect. The mean size of the KLA-modified TDNs was about 15 nm, and the TDNs were stable in FBS. The DOX loading efficiency of the TDNs was up to around 77%. The 3KLA-modified TDNs exhibited the most efficient DOX accumulation in mitochondria, leading to an effective release of cytochrome c, and the upregulated expression levels of caspase-9, caspase-3, p21 and p53. Meanwhile, 3KLA-TDNs/DOX elevated the pro-apoptotic Bax, reduced the anti-apoptotic Bcl-2 protein expression and increased the Bax/Bcl-2 ratio, which finally activated the mitochondria-mediated, programmed apoptosis pathway to enhance the anticancer efficacy in vitro. This 3KLA-TDN and DOX co-assembling strategy can be further developed to transport other anthracyclines and chemotherapeutic agents for enhanced apoptosis effects.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Adutos de DNA/farmacologia , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Mitocôndrias/efeitos dos fármacos , Nanoestruturas/química , Animais , Antibióticos Antineoplásicos/síntese química , Antibióticos Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Adutos de DNA/síntese química , Adutos de DNA/química , Relação Dose-Resposta a Droga , Doxorrubicina/síntese química , Doxorrubicina/química , Ensaios de Seleção de Medicamentos Antitumorais , Teste de Materiais , Camundongos , Estrutura Molecular , Células NIH 3T3 , Relação Estrutura-AtividadeRESUMO
The ATR protein kinase is known to protect cells from DNA damage induced during the replicative phase of the cell cycle. Small molecule ATR kinase inhibitors have therefore been developed to improve the effectiveness of DNA damage-based chemotherapy regimens aimed at killing rapidly proliferating tumor cells. However, whether ATR functions in a similar manner in non-replicating cells has not been examined and is important considering the fact that most cells in the body, including cancer stem cells in solid tumors, normally reside in either a quiescent or differentiated non-replicating state. Using cultured human cell lines maintained in a quiescent or slowly growing state in vitro, ATR was found to be activated following treatment with the common anti-cancer drug cisplatin in a manner dependent on the nucleotide excision repair (NER) system. Moreover, treatment with the ATR kinase inhibitors VE-821 and AZD6738 enhanced quiescent cell killing and apoptotic signaling induced by cisplatin. However, ATR kinase inhibition in quiescent cells treated with a low concentration of cisplatin also elevated the level of mutagenesis at the hypoxanthine phosphoribosyltransferase locus and resulted in increased levels of PCNA mono-ubiquitination. These results suggest that the excision gaps generated by NER may require a greater utilization of potentially mutagenic translesion synthesis polymerases in the absence of ATR kinase function. Thus, though ATR kinase inhibitors can aid in the killing of cisplatin-treated quiescent cells, such treatments may also result in a greater reliance on alternative mutagenic DNA polymerases to complete the repair of cisplatin-DNA adducts.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Mutagênese/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Adutos de DNA/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Humanos , Indóis , Morfolinas , Pirazinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas , Sulfonas/farmacologia , Sulfóxidos/farmacologiaRESUMO
Murine Krebs-2 tumor-initiating stem cells are known to natively internalize extracellular double-stranded DNA fragments. Being internalized, these fragments interfere in the repair of chemically induced interstrand cross-links. In the current investigation, 756 bp polymerase chain reaction (PCR) product containing bulky photoreactive dC adduct was used as extracellular DNA. This adduct was shown to inhibit the cellular system of nucleotide excision repair while being resistant to excision by this DNA repair system. The basic parameters for this DNA probe internalization by the murine Krebs-2 tumor cells were characterized. Being incubated under regular conditions (60 min, 24°C, 500 µL of the incubation medium, in the dark), 0.35% ± 0.18% of the Krebs-2 ascites cells were shown to natively internalize modified DNA. The saturating amount of the modified DNA was detected to be 0.37 µg per 106 cells. For the similar unmodified DNA fragments, this ratio is 0.73 µg per 106 cells. Krebs-2 tumor cells were shown to be saturated internalizing either (190 ± 40) × 103 molecules of modified DNA or (1,000 ± 100) × 103 molecules of native DNA. On internalization, the fragments of DNA undergo partial and nonuniform hydrolysis of 3' ends followed by circularization. The degree of hydrolysis, assessed by sequencing of several clones with the insertion of specific PCR product, was 30-60 nucleotides.
Assuntos
Carcinoma/genética , Adutos de DNA/genética , Fragmentação do DNA , DNA/genética , Animais , Carcinoma/patologia , Linhagem Celular Tumoral , DNA/farmacologia , Adutos de DNA/farmacologia , Reparo do DNA/efeitos dos fármacos , Humanos , CamundongosRESUMO
Developing a highly efficient carrier for tumor-targeted delivery and site-specific release of anticancer drugs is a good way to overcome the side effects of traditional cancer chemotherapy. Benefiting from the nontoxic and biocompatible characteristics, DNA-based drug carriers have attracted increasing attention. Herein, we reported a novel and readily manipulated strategy to construct spherical DNA nanocarriers. In this strategy, terminal deoxynucleotidyl transferase (TdT)-catalyzed DNA extension reaction is used to prepare a thick DNA layer on a gold nanoparticle (AuNP) surface by extending long poly(C) sequences from DNA primers immobilized on AuNPs. The poly(C) extension products can then hybridize with G-rich oligonucleotides to give CG-rich DNA duplexes (for loading anticancer drug doxorubicin, Dox) and multiple AS1411 aptamers. Via synergic recognition of multiple aptamer units to nucleolin proteins, biomarker of malignant tumors, Dox-loaded DNA carrier can be efficiently internalized in cancer cells and achieve burst release of drugs in acidic organelles because of i-motif formation-induced DNA duplex destruction. An as-prepared pH-responsive drug carrier was demonstrated to be promising for highly efficient delivery of Dox and selective killing of cancer cells in both in vitro and in vivo experiments, thus showing a huge potential in anticancer therapy.
Assuntos
Adutos de DNA , DNA Nucleotidilexotransferase/química , Doxorrubicina , Ouro , Nanopartículas Metálicas , Neoplasias Experimentais/tratamento farmacológico , Animais , Aptâmeros de Nucleotídeos , Adutos de DNA/química , Adutos de DNA/farmacologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Doxorrubicina/química , Doxorrubicina/farmacologia , Feminino , Ouro/química , Ouro/farmacologia , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células NIH 3T3 , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
ε-Poly-l-lysine (PL) has high antimicrobial activity and a wide antimicrobial spectrum and is applied broadly in the food industry. However, PL may lose part of its activity in phosphate systems, which are typically used in seafood processing. To enhance antimicrobial activity under high phosphate conditions, DNA/PL nanoparticles were fabricated via the nanoprecipitation method. The DNA/PL nanoparticle size was smallest when the ethanol to water ratio was 7:1, and the mean diameter was 101â¯nm. UV-vis showed hypochromism and a redshift of the DNA in the presence of PL, indicating an intercalative interaction between DNA and PL. FTIR spectroscopy revealed that strong hydrogen bonds and hydrophobic interactions were involved in DNA/PL nanoparticle formation. Compared with that of PL, the antimicrobial activity of the nanoparticles against S. aureus, B. subtilis, and E. coli was enhanced. Additionally, the DNA/PL nanoparticles still retained higher antimicrobial activity in the phosphate system than free PL. The antimicrobial mechanistic analysis provided evidence that the DNA/PL nanoparticles showed high bioactivity due to cell membrane damage. This work provides a potential method to enhance the antimicrobial activity of PL under adverse conditions, which can promote the application of PL in seafood containing phosphate compounds.
Assuntos
Antibacterianos , Bactérias/crescimento & desenvolvimento , Adutos de DNA , Lisina/análogos & derivados , Nanopartículas/química , Fosfatos/química , Polilisina , Antibacterianos/química , Antibacterianos/farmacologia , Adutos de DNA/química , Adutos de DNA/farmacologia , Lisina/química , Lisina/farmacologia , Polilisina/química , Polilisina/farmacologiaRESUMO
Despite the great progress in the field of drug delivery systems for cancer treatment over the last decade, many challenges still lie ahead, such as low drug loading, deep penetration of tumors, side effects, and the development of drug resistance. A class of cationic membrane lytic peptides has shown potential as an anticancer agent by inducing cancer cell death via membrane disruption; meanwhile, their intrinsic selectivity renders them as having low cytotoxicity towards noncancerous cells. Here, we report the use of a cationic peptide amphiphile (PA), named PAH6, to load doxorubicin (Dox) that is intercalated in an ATP-binding aptamer-incorporated DNA scaffold. The PA contains a cationic lytic sequence, (KLAKLAK)2, a polyhistidine segment for the "proton sponge" effect, and a hydrophobic alkyl tail to drive the self-assembly. Dox-loaded DNA was found to form a spherical nanocomplex (NC) with PAH6 with particle sizes below 100 nm at various ratios. Since the carrier PAH6 is also a therapeutic agent, the drug loadings of the NC reached up to ~86% within the ratios we tested, and Dox was released from the NC in an ATP-rich environment. In vitro studies indicate that the presence of PAH6 could permeabilize cell membranes and kill cells through fast membrane disruption and depolarization of mitochondrial membranes. The cytotoxicity tests were conducted using A549 nonsmall cell lung cancer cells and NIH-3T3 fibroblast cells. PAH6 showed selectivity towards A549 cells. Significantly, the Dox-DNA/PAH6 NC exhibited a synergistic effect against A549 cells, with the IC50 decreased up to ~90% for Dox and ~69% for PAH6 when compared to the IC50 values of the two components, respectively. Furthermore, the selectivity of PAH6 conferred to the complex an improved therapeutic index between A549 and NIH-3T3 cells. A 3D-cultured A549 spheroid model was adopted to test the capability of Dox-DNA/PAH6 for tumor penetration. The PAH6 or Dox-DNA/PAH6 complex was found to break the spheroids into pieces, while Dox-treated spheroids maintained their shapes. In summary, this work provides a new strategy for constructing nanomedicines using therapeutic agents to meet the features required by anticancer treatment.
Assuntos
Trifosfato de Adenosina , Antineoplásicos/farmacologia , Portadores de Fármacos/química , Peptídeos/química , Tensoativos/química , Trifosfato de Adenosina/química , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Aptâmeros de Nucleotídeos/química , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Adutos de DNA/química , Adutos de DNA/farmacologia , Doxorrubicina/química , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Sinergismo Farmacológico , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Nanopartículas/química , Tamanho da Partícula , Esferoides Celulares , Índice Terapêutico , Células Tumorais CultivadasRESUMO
Cisplatin is a major cancer chemotherapeutic drug. It kills cancer cells by damaging their DNA, mainly in the form of Pt-d(GpG) diadducts. However, it also has serious side effects, including nephrotoxicity and hepatotoxicity that limit its usefulness. Chronotherapy is taking circadian time into account during therapy to improve the therapeutic index, by improving efficacy and/or limiting toxicity. To this end, we tested the impact of clock time on excision repair of cisplatin-induced DNA damage at single-nucleotide resolution across the genome in mouse kidney and liver. We found that genome repair is controlled by two circadian programs. Repair of the transcribed strand (TS) of active, circadian-controlled genes is dictated by each gene's phase of transcription, which falls across the circadian cycle with prominent peaks at dawn and dusk. In contrast, repair of the nontranscribed strand (NTS) of all genes, repair of intergenic DNA, and global repair overall peaks at Zeitgeber time ZT08, as basal repair capacity, which is controlled by the circadian clock, peaks at this circadian time. Consequently, the TS and NTS of many genes are repaired out of phase. As most cancers are thought to have defective circadian rhythms, these results suggest that future research on timed dosage of cisplatin could potentially reduce damage to healthy tissue and improve its therapeutic index.
Assuntos
Antineoplásicos/farmacologia , Ritmo Circadiano/genética , Cisplatino/farmacologia , Adutos de DNA/farmacologia , Dano ao DNA , Reparo do DNA , Genoma Humano , Neoplasias/genética , Animais , Ritmo Circadiano/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Transcrição Gênica/efeitos dos fármacosRESUMO
The tobacco-derived nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are known human carcinogens. Following metabolic activation, NNK and NNN can induce a number of DNA lesions, including several 4-(3-pyridyl)-4-oxobut-1-yl (POB) adducts. However, it remains unclear to what extent these lesions affect the efficiency and accuracy of DNA replication and how their replicative bypass is influenced by translesion synthesis (TLS) DNA polymerases. In this study, we investigated the effects of three stable POB DNA adducts (O2-POB-dT, O4-POB-dT, and O6-POB-dG) on the efficiency and fidelity of DNA replication in HEK293T human cells. We found that, when situated in a double-stranded plasmid, O2-POB-dT and O4-POB-dT moderately blocked DNA replication and induced exclusively TâA (â¼14.9%) and TâC (â¼35.2%) mutations, respectively. On the other hand, O6-POB-dG slightly impeded DNA replication, and this lesion elicited primarily the GâA transition (â¼75%) together with a low frequency of the GâT transversion (â¼3%). By conducting replication studies in isogenic cells in which specific TLS DNA polymerases (Pols) were deleted by CRISPR-Cas9 genome editing, we observed that multiple TLS Pols, especially Pol η and Pol ζ, are involved in bypassing these lesions. Our findings reveal the cytotoxic and mutagenic properties of specific POB DNA adducts and unravel the roles of several TLS polymerases in the replicative bypass of these adducts in human cells. Together, these results provide important new knowledge about the biological consequences of POB adducts.
Assuntos
Carcinógenos/toxicidade , Adutos de DNA/farmacologia , Replicação do DNA/efeitos dos fármacos , Nicotiana/química , Nitrosaminas/química , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Células HEK293 , Humanos , Mutagênicos/efeitos adversos , Nitrosaminas/efeitos adversosRESUMO
Selective targeting is a crucial property of nanocarriers used for drug delivery in cancer therapy. We generated biotinylated octahedral DNA nanocages functionalized with folic acid through bio-orthogonal conjugation chemistry. Molecular modelling indicated that a distance of about 2.5 nm between folic acid and DNA nanocage avoids steric hindrance with the folate receptor. HeLa cells, a folate receptor positive tumour cell line, internalize folate-DNA nanocages with efficiency greater than 40 times compared to cells not expressing the folate receptors. Functionalized DNA nanocages are highly stable, not cytotoxic and can be efficiently loaded with the chemotherapeutic agent doxorubicin. After entry into cells, doxorubicin-loaded nanoparticles are confined in vesicular structures, indicating that DNA nanocages traffic through the endocytic pathway. Doxorubicin release from loaded DNA cages, facilitated by low pH of endocytic vesicles, induces toxic pathways that, besides selectively killing folate receptor-positive cancer cells, leads to cage degradation avoiding nanoparticles accumulation inside cells.