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Three artificial proteins that bind the gadolinium ion (Gd3+) with tumour-specific ligands were de novo engineered and tested as candidate drugs for binary radiotherapy (BRT) and contrast agents for magnetic resonance imaging (MRI). Gd3+-binding modules were derived from calmodulin. They were joined with elastin-like polypeptide (ELP) repeats from human elastin to form the four-centre Gd3+-binding domain (4MBS-domain) that further was combined with F3 peptide (a ligand of nucleolin, a tumour marker) to form the F3-W4 block. The F3-W4 block was taken alone (E2-13W4 protein), as two repeats (E1-W8) and as three repeats (E1-W12). Each protein was supplemented with three copies of the RGD motif (a ligand of integrin αvß3) and green fluorescent protein (GFP). In contrast to Magnevist (a Gd-containing contrast agent), the proteins exhibited three to four times higher accumulation in U87MG glioma and A375 melanoma cell lines than in normal fibroblasts. The proteins remained for >24 h in tumours induced by Ca755 adenocarcinoma in C57BL/6 mice. They exhibited stability towards blood proteases and only accumulated in the liver and kidney. The technological advantages of using the engineered proteins as a basis for developing efficient and non-toxic agents for early diagnosis of tumours by MRI as well as part of BRT were demonstrated.
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Elastina , Gadolínio , Animais , Meios de Contraste , Elastina/química , Gadolínio/química , Ligantes , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos , Proteínas RecombinantesRESUMO
Herein, we describe the design, synthesis, and biological evaluation of novel betulin and N-acetyl-d-galactosamine (GalNAc) glycoconjugates and suggest them as targeted agents against hepatocellular carcinoma. We prepared six conjugates derived via the C-3 and C-28 positions of betulin with one or two saccharide ligands. These molecules demonstrate high affinity to the asialoglycoprotein receptor (ASGPR) of hepatocytes assessed by in silico modeling and surface plasmon resonance tests. Cytotoxicity studies in vitro revealed a bivalent conjugate with moderate activity, selectivity of action, and cytostatic properties against hepatocellular carcinoma cells HepG2. An additional investigation confirmed the specific engagement with HepG2 cells by the enhanced generation of reactive oxygen species. Stability tests demonstrated its lability to acidic media and to intracellular enzymes. Therefore, the selected bivalent conjugate represents a new potential agent targeted against hepatocellular carcinoma. Further extensive studies of the cellular uptake in vitro and the real-time microdistribution in the murine liver in vivo for fluorescent dye-labeled analogue showed its selective internalization into hepatocytes due to the presence of GalNAc ligand in comparison with reference compounds. The betulin and GalNAc glycoconjugates can therefore be considered as a new strategy for developing therapeutic agents based on natural triterpenoids.
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Acetilgalactosamina/química , Antineoplásicos/farmacologia , Receptor de Asialoglicoproteína/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Triterpenos/química , Antineoplásicos/química , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Corantes Fluorescentes/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Ressonância de Plasmônio de SuperfícieRESUMO
Objective The problem of drug resistance to BRAF-targeted therapy often occurs in melanoma treatment. Activation of PI3K/AKT/mTOR signaling pathway is one of the mechanisms of acquired resistance and a potential target for treatment. In the current research, we investigated that dual inhibition of mTOR and MEK synergistically reduced the viability of melanoma cells in vitro. Methods A combination of rapamycin (a macrolide immunosuppressant, mTOR inhibitor) and binimetinib (an anti-cancer small molecule, selective inhibitor of MEK) was studied using a panel of melanoma cell lines, including patient-derived cells. Results It was found, that combinatorial therapy of rapamycin (250 nM) and binimetinib (2 µM) resulted in 25% of cell viability compared to either rapamycin (85%) or binimetinib alone (50%) for A375 and vemurafenib-resistant Mel IL/R cells. The suppressed activation of mTOR and MEK by combined rapamycin and binimetinib treatment was confirmed using Western blot assay. Cell death occured via the apoptosis pathway; however, the combination treatment significantly increased the apoptosis only for Mel IL/R cells. The enhanced cytotoxic effect was also associated with enhanced cell cycle arrest in the G0/G1 phase. Conclusion In general, we provide the evidence that dual inhibition of mTOR and MEK could be promising for further preclinical investigations.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzimidazóis/farmacologia , Melanoma/tratamento farmacológico , Sirolimo/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Apoptose/efeitos dos fármacos , Benzimidazóis/administração & dosagem , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sirolimo/administração & dosagem , Serina-Treonina Quinases TOR/antagonistas & inibidores , Vemurafenib/farmacologiaRESUMO
In vivo monitoring of reactive oxygen species (ROS) in tumors during treatment with anticancer therapy is important for understanding the mechanism of action and in the design of new anticancer drugs. In this work, a platinized nanoelectrode is placed into a single cell for detection of the ROS signal, and drug-induced ROS production is then recorded. The main advantages of this method are the short incubation time with the drug and its high sensitivity which allows the detection of low intracellular ROS concentrations. We have shown that our new method can measure the ROS response to chemotherapy in tumor-bearing mice in real-time. ROS levels were measured in vivo inside the tumor at different depths in response to doxorubicin. This work provides an effective new approach for the measurement of intracellular ROS by platinized nanoelectrodes.
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Antineoplásicos/farmacologia , Técnicas Biossensoriais , Doxorrubicina/farmacologia , Técnicas Eletroquímicas , Espécies Reativas de Oxigênio/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Neoplasias Experimentais/diagnóstico , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Células PC-3 , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismoRESUMO
Patients with metastatic melanoma are difficult to treat and have a very poor prognosis because of high resistance to therapy. Recent evidence indicates that tumors could overcome death through autophagy, a survival mechanism, which cancer cells use under lack of energy and nutrient deprivation. Melanoma cells have different sensitivity to temozolomide (TMZ) treatment. In this study, we showed that the combination of autophagy inhibitors chloroquine or LY294002 and TMZ induced enhanced cytotoxicity of alkylating agents on human melanoma cell lines. All assays were performed on patient-derived melanoma cell lines. The effectiveness of the combined treatment of TMZ and autophagy inhibitors was determined using an MTT assay. Next, we analyzed the expression mRNA level of Beclin 1, LC3B, and p62/STSQM1 and the relative expression of LC3B protein under combined treatment. Autophagic flux was determined by analysis of colocalization of Lysotracker Red and LC3B puncta. Apoptosis was measured by Annexin V/PI staining. Cell cycle analyses were carried out by flow cytometry. We showed that autophagy inhibition could enhance melanoma cell death combined with TMZ therapy. Chloroquine synergistically enhanced the TMZ-induced growth arrest and increased the G0/G1 population in Mel Z and Mel IL cell lines, but not Mel MTP. The expression analysis showed that autophagy involvement in TMZ enhanced cytotoxicity. Furthermore, LY294002, an early-stage autophagy, and PI3K inhibitor were found to exert similar effects. Both chloroquine and LY294002 improved the cytotoxic effect of TMZ treatment, making this combination applicable as a potent antitumor treatment for metastatic melanoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cloroquina/farmacologia , Cromonas/farmacologia , Dacarbazina/análogos & derivados , Melanoma/tratamento farmacológico , Morfolinas/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cloroquina/administração & dosagem , Cromonas/administração & dosagem , Dacarbazina/administração & dosagem , Dacarbazina/farmacologia , Sinergismo Farmacológico , Humanos , Melanoma/patologia , Morfolinas/administração & dosagem , Neoplasias Cutâneas/patologia , TemozolomidaRESUMO
BACKGROUND: Circulating tumor DNA (ctDNA) holds great potential for cancer therapy and can provide diagnostic and prognostic information before and during treatment. METHODS: Plasma DNA samples from 97 melanoma patients, 20 healthy donors and 3 patients with benign skin tumors were analyzed by microarray analysis and droplet digital PCR (ddPCR). RESULTS: A microarray for simultaneous detection of six BRAF V600 mutations in ctDNA has been developed. The method allows the detection of 0.05% mutated DNA from WT DNA background. For paired samples (pre-surgery plasma and tumor tissue) isolated from 74 patients, the concordance of genotypes between tumor DNA and ctDNA was 65% (48/74). BRAF mutations in ctDNA were detected in 27/50 patients with BRAF-positive tumors and in 3/24 patients with BRAF wild-type tumors. The presence of ctDNA BRAF mutations in 23 plasma samples from melanoma patients undergoing therapy correlated significantly with tumor progression (P=0.005). The increase in cell-free DNA levels measured by ddPCR also correlated with disease progression (P=0.02). The concordance of results obtained by microarray identification of BRAF mutations and those obtained by ddPCR was 91%. CONCLUSION: The novel microarray-based approach can be a useful non-invasive tool for accurate identification of ctDNA BRAF mutations to monitor disease progression.
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DNA Tumoral Circulante/genética , Melanoma/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Estudos de Casos e Controles , Progressão da Doença , Feminino , Humanos , Biópsia Líquida , Masculino , Melanoma/sangue , Melanoma/patologia , Neoplasias Cutâneas/sangue , Neoplasias Cutâneas/patologiaRESUMO
The phenomenon of the primary or acquired resistance of cancer cells to antitumor drugs is among the key problems of oncology. For breast cancer, the phenomenon of the resistance to hormonal or target therapy may be based on the numerous mechanisms including the loss or mutation of estrogen receptor, alterations of antiapoptotic pathways, overexpression of growth-related signaling proteins, etc. The perspective approaches for overcoming the resistance may be based on the usage of compounds such as inhibitors of the cell energetic metabolism. Among the latter, the antidiabetic drug metformin exerts antitumor activity via the activation of AMPK and the subsequent inhibition of mTOR signaling. The experiments were performed on the ERα-positive MCF-7 breast cancer cells, the MCF-7 sublines resistant to tamoxifen (MCF-7/T) and rapamycin (MCF-7/Rap), and on triple-negative MDA-MB-231 breast cancer cells. We have demonstrated metformin's ability to enhance the cytostatic activity of the tamoxifen and rapamycin on both parent MCF-7 cells and MCF-7-resistant derivates mediated via the suppression of mTOR signaling and growth-related transcriptional factors. The cooperative effect of metformin and tested drugs was realized in an estrogen-independent manner, and, in the case of tamoxifen, was associated with the activation of apoptotic cell death. Similarly, the stimulation of apoptosis under metformin/tamoxifen co-treatment was shown to occur in the MCF-7 cells after steroid depletion as well as in the ERα-negative MDA-MB-231 cells. We conclude that metformin co-treatment may be used for the increase and partial restoration of the cancer cell sensitivity to hormonal and target drugs. Moreover, the combination of metformin with tamoxifen induces the apoptotic death in the ERα-negative breast cancer cells opening the additional perspectives in the treatment of estrogen-independent breast tumors.
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Oncolytic viruses, including live attenuated measles virus (MV) vaccine strains, have recently been shown as promising therapeutic agents against human malignancies. In this study, the oncolytic potential of the attenuated vaccine strain Leningrad-16 (L-16) of MV was evaluated in a panel of human metastatic melanoma cell lines. The L-16 measles virus was shown to replicate within melanoma cells mediating direct cell killing of tumor cells, although all melanoma cell lines varied in regard to their ability to respond to L-16 MV infection, as revealed by the different pattern of the Interferon Stimulated Gene expression, cytokine release and mechanisms of cell death. Furthermore, the statistically significant L-16 measles virus related tumor growth inhibition was demonstrated in a melanoma xenograft model. Therefore, L-16 MV represents an appealing oncolytic platform for target delivery of therapeutic genes along with other attenuated measles virus strains.
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Vírus do Sarampo/patogenicidade , Melanoma/terapia , Melanoma/virologia , Vírus Oncolíticos/patogenicidade , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Vacina contra Sarampo , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia Viral Oncolítica/métodos , Vacinas Atenuadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Flow cytometry using fluorescent antibodies (FC) is the method of choice for the quantitation of proteins expressed at the surface or inside the cell, but, however, does not allow to selectively measure nuclear expression. We therefore sought to develop a method for the extraction of intact cell nuclei, which can be used for their subsequent immunofluorescent analysis by FC. The studied protein was vascular endothelial growth factor-receptor-type 1 (VEGFR-1) which is important in tumor survival and metastasis. Two human cell lines, A431 (epidermoid carcinoma of skin with low invasive and metastatic potential) and BRO (highly aggressive amelanotic melanoma), were used as examples for tumor cells, and normal human fibroblasts PHF served as a control line. The quality of the extracted nuclei was assessed by their intactness and purity from cytoplasm. The high content of the nuclear markers (PCNA = proliferating cell nuclear antigen, lamin A/C) in the extracted nuclei with almost complete absence of the cytoplasmic ß-tubulin demonstrated that the protocol can be used to obtain a pure suspension of single intact cell nuclei. The measurement of the nuclear VEGFR-1 content revealed that it was present only in tumor cell nuclei and that in more malignant BRO cells the receptor content was 1.75 times higher than in A431 (p = 0.014). Thus, the developed method of extraction of cell nuclei for subsequent FC analysis is suitable for the quantitative evaluation of protein content in the native nucleus.
Assuntos
Núcleo Celular/metabolismo , Separação Celular/métodos , Citometria de Fluxo/métodos , Análise de Célula Única/métodos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/isolamento & purificação , Adulto , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Estudos de Viabilidade , Feminino , Fibroblastos , Imunofluorescência/métodos , Humanos , Masculino , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Melanoma is one of the most aggressive and treatment-resistant tumors that responsible for majority of skin-cancer related deaths. Here we propose a combination of MEK inhibitor binimetinib with metformin as a promising therapy against human melanoma cells in vitro, including BRAF -mutated A375, Mel Z, and Mel IL cells, and NRAS-mutated Mel MTP and Mel Me cells. Additionally, we obtained two close to clinical practice models of melanoma progression. The first one was vemurafenib-resistant Mel IL/R melanoma cells with acquired resistance to BRAF inhibition-targeted therapy, and the second one was tumor spheroids, which are 3D in vitro model of small-size solid tumors in vivo. The cytotoxicity of binimetinib and metformin was synergistic in both 2D and 3D melanoma culture and mediated through apoptotic pathway. The combination reduced the number of melanoma-formed colonies, inhibited cell invasion and migration, and led to G0/G1 cell cycle arrest through cyclin D/CDK4/CDK6 pathway. The mechanism of metformin and binimetinib synergy in melanoma cells was associated with increased activation of p-AMPKα and decreased p-ERK, but not with alterations in p-mTOR. In summary, the combination of metformin and binimetinib resulted in stronger anti-proliferative effects on melanoma cells compared to binimetinib alone, and therefore could be promising for clinical applications.
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Benzimidazóis/administração & dosagem , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Melanoma/enzimologia , Metformina/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Hipoglicemiantes/administração & dosagem , Melanoma/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagemRESUMO
The incidence of malignant melanoma is increasing. The discovery of agents specifically targeting the mutated cascades has provided a good response for patients with oncogenic B-Raf proto-оncogene, serine/threonine kinase (BRAF). However, numerous studies continue to focus on novel methods of treatment to overcome acquired resistance to novel drugs. Recently, it has been revealed that inhibition of endoplasmic reticulum (ER) stress chaperon 78 kDa glucose-regulated protein 78 (GRP78) leads to down-regulation of autophagy and increased sensitivity to temozolomide (TMZ) treatment. Melanoma cells have a different sensitivity to TMZ treatment, which corresponds to the basal autophagy level. In the present study, we demonstrated that downregulation of GRP78 mitigated chemoresistance to TMZ in three melanoma cell lines. We found that downregulation of GRP78 led to inhibition of autophagy, cell cycle arrest in the G0/G1 phase, and activation of caspase-7-induced apoptosis, and this was affected by the initial autophagy level. Moreover, inhibition of GRP78 mitigated the combined TMZ and chloroquine effect. Our data revealed that autophagy inhibition through downregulation of ER stress response could overcome resistance to TMZ treatment in melanoma cells with a high basal level of autophagy treatment, which makes this combination a potential potent antitumor treatment for metastatic melanoma.
Assuntos
Dacarbazina/análogos & derivados , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Melanoma/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Caspase 7/genética , Linhagem Celular Tumoral , Dacarbazina/administração & dosagem , Chaperona BiP do Retículo Endoplasmático , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melanoma/genética , Melanoma/patologia , Camundongos , Proteínas Proto-Oncogênicas B-raf/genética , Temozolomida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Target inhibitors are used for melanoma treatment, and their effectiveness depends on the tumor genotype. We developed a diagnostic biochip for the detection of 39 clinically relevant somatic mutations in the BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2 genes. We used multiplex locked nucleic acid (LNA) PCR clamp for the preferable amplification of mutated over wild type DNA. The amplified fragments were labeled via the incorporation of fluorescently labeled dUTP during PCR and were hybridized with specific oligonucleotides immobilized on a biochip. This approach could detect 0.5% of mutated DNA in the sample analyzed. The method was validated on 253 clinical samples and six melanoma cell lines. Among 253 melanomas, 129 (51.0%) BRAF, 45 (17.8%) NRAS, 6 (2.4%) KIT, 4 (1.6%) GNAQ, 2 (0.8%) GNA11, 2 (0.8%) MAP2K1 and no MAP2K2 gene mutations were detected by the biochip assay. The results were compared with Sanger sequencing, next generation sequencing and ARMS/Scorpion real-time PCR. The specimens with discordant results were subjected to LNA PCR clamp followed by sequencing. The results of this analysis were predominantly identical to the results obtained by the biochip assay. Infrequently, we identified rare somatic mutations. In the present study we demonstrate that the biochip-based assay can effectively detect somatic mutations in approximately 70% of melanoma patients, who may require specific targeted therapy.
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Alofanib (RPT835) is a novel selective allosteric inhibitor of fibroblast growth factor receptor 2 (FGFR2). We showed previously that alofanib could bind to the extracellular domain of FGFR2 and has an inhibitory effect on FGF2-induced phoshphorylation of FRS2α. In the present study, we further showed that alofanib inhibited phosphorylation of FRS2α with the half maximal inhibitory concentration (IC50) values of 7 and 9 nmol/l in cancer cells expressing different FGFR2 isoforms. In a panel of four cell lines representing several tumour types (triple-negative breast cancer, melanoma, and ovarian cancer), alofanib inhibited FGF-mediated proliferation with 50% growth inhibition (GI50) values of 16-370 nmol/l. Alofanib dose dependently inhibited the proliferation and migration of human and mouse endothelial cells (GI50 11-58 nmol/l) compared with brivanib and bevacizumab. Treatment with alofanib ablated experimental FGF-induced angiogenesis in vivo. In a FGFR-driven human tumour xenograft model, oral administration of alofanib was well tolerated and resulted in potent antitumour activity. Importantly, alofanib was effective in FGFR2-expressing models. These results show that alofanib is a potent FGFR2 inhibitor and provide strong rationale for its evaluation in patients with FGFR2-driven cancers.