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BACKGROUND: The mechanisms enabling dynamic shifts between drug-resistant and drug-sensitive states in cancer cells are still underexplored. This study investigated the role of targeted autophagic protein degradation in regulating ovarian cancer stem cell (CSC) fate decisions and chemo-resistance. METHODS: Autophagy levels were compared between CSC-enriched side population (SP) and non-SP cells (NSP) in multiple ovarian cancer cell lines using immunoblotting, immunofluorescence, and transmission electron microscopy. The impact of autophagy modulation on CSC markers and differentiation was assessed by flow cytometry, immunoblotting and qRT-PCR. In silico modeling and co-immunoprecipitation identified ID1 interacting proteins. Pharmacological and genetic approaches along with Annexin-PI assay, ChIP assay, western blotting, qRT-PCR and ICP-MS were used to evaluate effects on cisplatin sensitivity, apoptosis, SLC31A1 expression, promoter binding, and intracellular platinum accumulation in ID1 depleted backdrop. Patient-derived tumor spheroids were analyzed for autophagy and SLC31A1 levels. RESULTS: Ovarian CSCs exhibited increased basal autophagy compared to non-CSCs. Further autophagy stimulation by serum-starvation and chemical modes triggered proteolysis of the stemness regulator ID1, driving the differentiation of chemo-resistant CSCs into chemo-sensitive non-CSCs. In silico modeling predicted TCF12 as a potent ID1 interactor, which was validated by co-immunoprecipitation. ID1 depletion freed TCF12 to transactivate the cisplatin influx transporter SLC31A1, increasing intracellular cisplatin levels and cytotoxicity. Patient-derived tumor spheroids exhibited a functional association between autophagy, ID1, SLC31A1, and platinum sensitivity. CONCLUSIONS: This study reveals a novel autophagy-ID1-TCF12-SLC31A1 axis where targeted autophagic degradation of ID1 enables rapid remodeling of CSCs to reverse chemo-resistance. Modulating this pathway could counter drug resistance in ovarian cancer.
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Autofagia , Resistencia a Medicamentos Antineoplásicos , Proteína 1 Inibidora de Diferenciação , Células-Tronco Neoplásicas , Neoplasias Ovarianas , Humanos , Feminino , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Proteína 1 Inibidora de Diferenciação/metabolismo , Proteína 1 Inibidora de Diferenciação/genética , Linhagem Celular Tumoral , Cisplatino/farmacologiaRESUMO
Noninvasive, real-time, longitudinal imaging of protein functions in living systems with unprecedented specificity is one of the critical challenges of modern biomedical research. Toward that goal, here, we report a platform fusion technology called activity-based protein profiling-bioluminescence resonance energy transfer (ABPP-BRET). This method provides an opportunity to study the post-translational modification of a target protein in real time in living systems in a longitudinal manner. This semisynthetic BRET biosensor method is used for target engagement studies and further for inhibitor profiling in live cells. The simplicity of this method coupled with the critical physical distance-dependent BRET readout turned out to be a powerful method, thus pushing the activity-based protein profiling technology to the next level.
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Bioluminescence resonance energy transfer (BRET) is one of the most promising approaches used for noninvasive imaging of protein-protein interactions in vivo. Recently, our team has discovered a genetically encodable bioluminescent system from the fungus Neonothopanus nambi and identified a novel luciferase that represents an imaging tool orthogonal to other luciferin-luciferase systems. We demonstrated the possibility of using the fungal luciferase as a new BRET donor by creating fused pairs with acceptor red fluorescent proteins, of which tdTomato provided the highest BRET efficiency. Using this new BRET system, we also designed a mTOR pathway specific rapamycin biosensor by integrating the FRB and FKBP12 protein dimerization system. We demonstrated the specificity and efficacy of the new fungal luciferase-based BRET combination for application in mammalian cell culture that will provide the unique opportunity to perform multiplexed BRET assessment in the future.
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This study demonstrates the development of a humanized luciferase imaging reporter based on a recently discovered mushroom luciferase (Luz) from Neonothopanus nambi. In vitro and in vivo assessments showed that human-codon-optimized Luz (hLuz) has significantly higher activity than native Luz in various cancer cell types. The potential of hLuz in non-invasive bioluminescence imaging was demonstrated by human tumor xenografts subcutaneously and by the orthotopic lungs xenograft in immunocompromised mice. Luz enzyme or its unique 3OH-hispidin substrate was found to be non-cross-reacting with commonly used luciferase reporters such as Firefly (FLuc2), Renilla (RLuc), or nano-luciferase (NLuc). Based on this feature, a non-overlapping, multiplex luciferase assay using hLuz was envisioned to surpass the limitation of dual reporter assay. Multiplex reporter functionality was demonstrated by designing a new sensor construct to measure the NF-κB transcriptional activity using hLuz and utilized in conjunction with two available constructs, p53-NLuc and PIK3CA promoter-FLuc2. By expressing these constructs in the A2780 cell line, we unveiled a complex macromolecular regulation of high relevance in ovarian cancer. The assays performed elucidated the direct regulatory action of p53 or NF-κB on the PIK3CA promoter. However, only the multiplexed assessment revealed further complexities as stabilized p53 expression attenuates NF-κB transcriptional activity and thereby indirectly influences its regulation on the PIK3CA gene. Thus, this study suggests the importance of live cell multiplexed measurement of gene regulatory function using more than two luciferases to address more realistic situations in disease biology.
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Agaricales , Neoplasias Ovarianas , Humanos , Animais , Feminino , Camundongos , NF-kappa B , Linhagem Celular Tumoral , Proteína Supressora de Tumor p53/genética , Luciferases/genética , Agaricales/genética , Classe I de Fosfatidilinositol 3-QuinasesRESUMO
Among the EGFR family of receptors, HER3 is considered as a pseudo-kinase which primarily interacts with HER2 in presence of heregulin-1ß. We identified two hotspot mutations i.e. G284R and D297Y and one double mutant HER2-S310F/HER3-G284R in breast cancer patients. Long term MDS (7.5 µs) revealed that HER3-D297Y and HER2-S310F:HER3-G284R do not allow the interaction with HER2 as these mutations cause dramatic conformational changes in its flanking regions. This results in formation of an unstable HER2-WT:HER3-D297Y heterodimer, thereby abrogating the downstream signalling by AKT. We found that His228 and Ser300 of HER3-D297Y form stable interactions with Glu245 and Tyr270 of EGFR-WT, in the presence of either EGF or heregulin-1ß. Applying TRIM-ing mediated direct knockdown of endogenous EGFR protein, specificity of the unconventional EGFR:HER3-D297Y interaction was validated. Due to this unusual ligand mediated interaction, cancer cells were found susceptible to EGFR targeted therapeutics i.e. Gefitinib and Erlotinib. Further, in TCGA analysis, BC patients harbouring HER3-D297Y mutation showed increased p-EGFR levels as compared to the patients harbouring HER3-WT and HER3-G284R mutations. For the first time, this comprehensive study showed the importance of specific hotspot mutations in HER3 dimerization domain can defy the Trastuzumab therapy, rather cells become susceptible to the EGFR inhibitors.
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Neoplasias da Mama , Receptor ErbB-3 , Feminino , Humanos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Dimerização , Receptores ErbB/genética , Receptores ErbB/metabolismo , Mutação , Neuregulina-1/genética , Neuregulina-1/metabolismo , Neuregulina-1/farmacologia , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismoRESUMO
Photothermal therapy (PTT) has emerged as a very potent therapeutic approach in the treatment of tumors. Gold nanoparticles have gained considerable scientific interest as a photosensitizer due to their absorbance in the near-infrared regions. However, their biodegradation and excretion from the body is a challenge. Various biodegradable systems consisting of liposomes and polymers have been synthesized, but their precise manufacturing and decomposition mechanisms have not yet been explored. Using zein nanoparticles as a template, we have fabricated a glutathione-functionalized gold core shell type of formulation. The scalability of the one-step seedless gold coating process is also reported. The synthesis procedure of these tunable nanoparticles is understood with TEM. The thermal degradation of the material under the physiological conditions is thoroughly examined using UV and TEM. In vitro PTT effectiveness on breast cancer cells is assessed after an extensive in vitro toxicity research. The mechanism of cell death is studied using ROS and cell cycle analysis. The material exhibited good efficacy as a PTT agent in mice and showed non-toxicity up to 14 days. The renal clearance study of the material in mice shows its disintegration into renal clearable minute gold seeds. All the findings suggest biodegradable glutathione-functionalized gold core-shell nanoparticles as potential photothermal cancer treatment agents.
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Nanopartículas Metálicas , Nanopartículas , Fotoquimioterapia , Animais , Camundongos , Ouro/farmacologia , Ouro/uso terapêutico , Nanopartículas Metálicas/uso terapêutico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Fototerapia , Linhagem Celular TumoralRESUMO
A generalized therapeutic strategy for various disease conditions, including cancer, is to deplete or inactivate harmful protein targets. Various forms of protein or gene silencing molecules, e.g., small molecule inhibitors, RNA interference (RNAi), and microRNAs (miRNAs) have been used against druggable targets. Over the past few years, targeted protein degradation (TPD) approaches have been developed for direct degradation of candidate proteins. Among the TPD approaches, proteolysis targeting chimeras (PROTACs) have emerged as one of the most promising approaches for the selective elimination of proteins via the ubiquitin-proteasome system. Other than PROTACs, TPD methods with potential therapeutic use include intrabody-mediated protein knockdown and tripartite motif-21 (TRIM-21) mediated TRIM-Away. In this review, protein knockdown approaches, their modes of action, and their advantages over conventional gene knockdown approaches are summarized. In cancers, disease-associated protein functions are often executed by specific post-translational modifications (PTMs). The role of TRIM-Away is highlighted in the direct knockdown of PTM forms of target proteins. Moreover, the application challenges and the prospective clinical use of TPD approaches in various diseases are also discussed.
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Photothermal therapy (PTT) has emerged as a fast, precisive, and cost-effective anticancer therapy protocol. Here we applied our previously designed nanomaterial (Tocophotoxil) for prospective PTT application to manage radiation- and chemo-resistant cancers in a preclinical model. A PTT dose vs. efficacy relationship was established for radioresistant breast (ZR-75-1 50Gy, 4T1 20Gy) and chemo-resistant ovarian (A2780LR) cancer cells and tumors in mice models. Compared to the sensitive cases, resistant cells treated with PTT for a shorter duration show higher endurance. However, preclinical tumor xenografts treated with optimal PTT dose show 2-3 fold higher longevity (P ≤ 0.05) of treated mice monitored by non-invasive imaging methods. Elevated ERK and AKT activation in radioresistant or only AKT activation in chemo-resistant cells were contributory to higher cell survival in sub-optimal PTT dose. A comprehensive single-cell Raman map of PTT treated ZR-75-1 cell reveals broad-spectrum macromolecular deformities, including protein damage features. Marked induction of pJNK, unfolded protein response (UPR) pathway, increased reactive oxygen species (ROS), and lipid peroxidation in PTT-treated cells disrupted the intracellular homeostasis. Analyzing cellular ultrastructure, the coexistence of swollen endoplasmic reticulum, and autophagic bodies after PTT indicate possible coordination between UPR and autophagy pathways. Therefore, this comprehensive study provides new evidence on the potential impact of PTT as a standalone therapy for ablation of failed conventional therapy-resistant cancers in vivo, the success of which is intricately linked to the PTT dose optimization. The study, for the first time, also illustrates that under PTT treatment, concerted action of novel molecular switches such as JNK activation and UPR activation plays a vital role in triggering autophagy and cancer cell death.
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Neoplasias , Terapia Fototérmica , Humanos , Animais , Camundongos , Proteínas Proto-Oncogênicas c-akt , Estudos Prospectivos , Camundongos Endogâmicos BALB C , Neoplasias/terapiaRESUMO
Proteins play an important part in almost all life activities and across all organisms. Proteins occasionally act on their own but rather fulfill most of their biological tasks by cooperating with other proteins or ligand molecules. The bioluminescence resonance energy transfer (BRET) assay serves to measure dynamic events such as protein-protein or protein-ligand interactions in vitro or in-vivo. With several inherent attributes such as rapid and fairly sensitive ratio-metric measurements, assessment of interactions irrespective of protein location within the cellular compartment, cost-effectiveness consenting to high-throughput screening compatibility, makes BRET a popular genetic reporter-based assay system for protein-protein interaction (PPI) studies. Based on the Förster principle, BRET allows to judge if the proximity has been achieved between the interacting partners. In recent years, the BRET application has emerged as a significantly versatile assay format by using multiple detection devices such as a plate reader or in-vivo optical imaging platform, or even a bioluminescence microscope has expanded its scope for advancing PPI studies. Beyond the scope of quantitative measurement of PPIs, molecular optical imaging applications based on BRET assay have expanded the scope for screening pharmacological compounds by unifying live cell and in-vivo animal-/plant-based experiments using the same platform technology. In this chapter, we have given intricate methodological details for performing in-vitro and in-vivo BRET experiments, primarily by using donor/acceptor reporter protein combinations.
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Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Medições Luminescentes , Animais , Bioensaio , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Transferência de Energia , Ligantes , Medições Luminescentes/métodos , ProteínasRESUMO
PURPOSE: In this work for the first time, we showed specific and direct knockdown of important oncogenic proteins of interest and their phospho-PTM targets in tripartite motif containing-21 (TRIM21) overexpressing breast cancer (BC) cells. We revealed the functional and therapeutic consequences of this protein knockdown approach called 'TRIM-ing'. METHODS: To target HER2, HER3, STAT3 or their activated forms, electroporation and puls-in transfection were standardized for mAb delivery in AU565 and MCF7 BC cell lines. Cancer cells were treated with HER2-targeted medicines (Trastuzumab and Neratinib) or STAT3 targeted inhibitors (Stattic and Niclosamide) with or without respective target TRIM-ing. Real-time PCR, immunoblotting, immunofluorescence, cytotoxicity, short- and long-term cell survival assessments were done following standard methodologies. 3-D structure modelling was used to verify the binding of mAb onto the STAT3 target. RESULTS: TRIM-ing of HER2 or HER3 receptors or their activated phospho-forms in BC cells showed rapid degradation of respective protein forms, shattering down the downstream signaling (p-ERK, p-AKT) that lasts for up to 7-8 days. This significantly inhibited BC survival (p < 0.001), showing a synergistic therapeutic effect with HER2 medicine trastuzumab or neratinib. Additionally, specific TRIM-ing ability of canonical pY705 or non-canonical pS727 PTMs of STAT3 protein was demonstrated in MCF7 cells, causing significant cytotoxicity (p < 0.05). TRIM-ing of STAT3 PTM, when combined with the same PTM-specific inhibitors, a synergistic treatment effect was observed. CONCLUSION: The work demonstrated that TRIM-ing could directly reduce various oncogenic targets or their specific activated form inside the cancer cells without compensatory pathway activation, a conundrum limiting the therapeutic benefit of current personalized medicines.
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Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Receptor ErbB-2/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Niclosamida/uso terapêutico , Medicina de Precisão , Resistencia a Medicamentos Antineoplásicos , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Linhagem Celular TumoralRESUMO
On the basis of the boron neutron capture therapy (BNCT) modality, we have designed and synthesized a zinc gallate (ZnGa2O4)-based nanoformulation for developing an innovative theranostic approach for cancer treatment. Initially, the (ZnGa1.995Cr0.005O4 or ZnGa2O4:(0.5%)Cr persistent luminescence nanoparticles (PLNPs) embedded on silica matrix were synthesized. Their surface functionalization was performed using organic synthesis strategies to attach the amine functional moieties which were further coupled with poly(vicinal diol). These diols were helpful for conjugation with 10B(OH)3, which subsequently served to couple with an in-house-synthesized variant of pH-(low)-insertion peptide (pHLIP) finally giving a tumor-targeting nanoformulation. Most importantly, the polymeric diols helped in conjugation of a substantial number of 10B to provide the therapeutic dose required for effective BNCT. This nanoformulation internalized substantially (â¼80%) to WEHI-164 cancer cells within 6 h. Tumor homing studies indicated that the accumulation of this formulation at the acidic tumor site was within 2 h. The in vitro evaluation of the formulation against WEHI-164 cancer cells followed by neutron irradiation revealed its potent cytotoxicity with IC50 â¼ 25 µM. In the case of studies on animal models, the melanoma-induced C57BL/6 and fibrosarcoma-induced BALB/c mice were treated with formulations through intratumoral and intravenous injections, respectively, followed by neutron irradiation, leading to a significant killing of the cancer cells, which was evidenced by a reduction in tumor volume (75-80%) as compared with a control tumor. Furthermore, the histopathological studies confirmed a damaging effect only on tumor cells, while there was no sign of damage to the vital organs in treated mice as well as in controls.
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Terapia por Captura de Nêutron de Boro , Melanoma , Nanopartículas , Animais , Luminescência , Camundongos , Camundongos Endogâmicos C57BL , ZincoRESUMO
Recurrent metastatic epithelial ovarian cancer (EOC) is challenging and associated with treatment limitations, as the mechanisms governing the metastatic behavior of chemoresistant EOC cells remain elusive. Using orthotopic xenograft mouse models of sensitive and acquired platinum-taxol-resistant A2780 EOC cells, we studied the mechanistic role of insulin like growth factor 1 receptor (IGF1R) signaling in the regulation of organ-specific metastasis of EOC cells undergoing acquirement of chemoresistance. Biochemical assays and organ-specific fibroblast-EOC cell co-culture were used to study the differential metastatic characteristics of sensitive vs. chemoresistant EOC cells, and the key molecule/s underlying the organ-specific homing of chemoresistant EOC cells were identified through subtractive LC/MS profiling of the co-culture secretome. The role of the identified molecule was validated through genetic/pharmacologic perturbation experiments. Acquired chemoresistance augmented organ-specific metastasis of EOC cells and enhanced lung homing, particularly for the late-stage chemoresistant cells, which was abrogated after IGF1R silencing. Escalation of chemoresistance (intrinsic and acquired) conferred EOC cells with higher adhesion toward primary lung fibroblasts, largely governed by the α6 integrin-IGF1R dual signaling axes. Subtractive analysis of the co-culture secretome revealed that interaction with lung fibroblasts induced the secretion of S100A4 from highly resistant EOC cells, which reciprocally activated lung fibroblasts. Genetic and pharmacologic inhibition of S100A4 significantly lowered distant metastases and completely abrogated lung-tropic nature of late-stage chemoresistant EOC cells. These results indicate that chemoresistance exacerbates organ-specific metastasis of EOC cells via the IGF1R-α6 integrin-S100A4 molecular network, of which S100A4 may serve as a potential target for the treatment of recurrent metastatic EOC.
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Carcinoma Epitelial do Ovário/tratamento farmacológico , Integrina alfa6/genética , Receptor IGF Tipo 1/genética , Proteína A4 de Ligação a Cálcio da Família S100/genética , Animais , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Xenoenxertos , Humanos , Camundongos , Metástase Neoplásica , Paclitaxel/farmacologia , Platina/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Therapy induced rewiring of signalling networks often lead to acquirement of platinum-resistance, thereby necessitating the use of non-platinum agents as second-line treatment particularly for epithelial ovarian cancer (EOC). A prior subject-specific assessment can guide the choice of optimal non-platinum agent/s and possible targeted therapeutic/s. Assessment of protein-protein interactions are superior to simple cytotoxicity assays to determine therapeutic efficacy and associated molecular responses. Utilizing improved PIP3-AKT and ERK1/2 activation Bioluminescence Resonance Energy Transfer (BRET) sensors, we report chemotherapy-induced ERK1/2 activation predominantly in cisplatin-paclitaxel resistant EOC cells and increased activation of both ERK1/2 and AKT in malignant ascites derived cancer cells from platinum-resistant patients but not from treatment-naive or platinum-sensitive relapse patients. Further, majority of the non-platinum drugs except irinotecan increased ERK1/2 activation in platinum-taxol resistant cells as observed by live-cell BRET assessment which were associated with p90RSK1/2 and BAD activation along with upregulation of multidrug transporter gene ABCC1 and cell survival genes like cyclin D1 and Bcl2. Interestingly, only irinotecan was able to sensitize these resistant cells. Altogether, this first report of BRET based sensing of molecular pathway activations in platinum resistant cell lines and patient's derived cancer cells highlight the clinical potential of BRET sensors in management of therapy resistant cancer.
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Photothermal-therapy (PTT) inculcates near-infrared laser guided local heating effect, where high degree of precision is expected, but not well proven to-date. An ex vivo tissue biochemical map with molecular/biochemical response showing the coverage area out of an optimized PTT procedure can reveal precision information. In this work, Raman-microscopic mapping and linear discriminant analysis of spectra of PTT treated and surrounding tissue areas ex vivo are done, revealing three distinct spectral clusters/zones, with minimal overlap between the core treated and adjacent untreated zone. The core treated zone showed intense nucleic-acid, cytochrome/mitochondria and protein damage, an adjacent zone showed lesser degree of damages and far zone showed minimal/no damage. Immunohistochemistry for γH2AX (DNA damage marker protein) in PTT exposed tissue also revealed similar results. Altogether, this study reveals the utility of Raman-microspectroscopy for fine-tuning safety parameters and precision that can be achieved from PTT mediated tumor ablation in preclinical/clinical application.
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Nanopartículas Metálicas/química , Neoplasias/terapia , Terapia Fototérmica/métodos , Nanomedicina Teranóstica/tendências , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Ouro/química , Ouro/farmacologia , Histonas/genética , Humanos , Neoplasias/genética , Neoplasias/patologia , Análise Espectral RamanRESUMO
The enzyme cholesteryl ester transfer protein (CETP), involved in cholesterol metabolism and transportation, is one of the main causes of cardiovascular (CV) disease (CVD). When the CETP concentration is decreased by CETP inhibitors (e.g., anacetrapib, torcetrapib, obicetrapib, etc.), high-density lipoprotein (HDL) particles are formed and low-density lipoprotein (LDL) is decreased along with cholesterol transportation alteration, which reduces the development of atherosclerosis. Here, we discuss the role of CETP inhibitors in reducing well-known 'bad' cholesterols and the current status of trials of different CETP inhibitors, their adverse effects, and limitations, as well as the pathophysiology of CETP.
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Doenças Cardiovasculares/prevenção & controle , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Animais , Doenças Cardiovasculares/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , HumanosRESUMO
Niclosamide (Nic), an FDA approved antihelminthic drug, is being repurposed as a potent anti-cancer and anti-inflammatory agent. Niclosamide exhibits anti-cancer activity in multiple cancer types, including breast, colon, and prostate cancers. Niclosamide, a BCS II drug, is practically insoluble in water and sparingly soluble in organic solvents (ethanol, dimethyl sulfoxide), leading to limited therapeutic applications, and necessitates the need for a drug carrier. Herein, we report the preparation of polydopamine nanoparticles loaded with niclosamide (Nic-PDA NPs). The designed formulation had a very high loading efficiency (~30%) and entrapment efficiency close to 90%. The average hydrodynamic diameter of Nic-PDA NPs was 146.3 nm, with a narrow size distribution (PDI = 0.039). The formulation exhibited a pH-dependent drug release profile, with ~35% drug released at pH 7.4 after 120 h, compared to > 50% at pH 5.5 in simulated physiological conditions. The NPs exhibited time-dependent cellular uptake and were primarily localized in the cytoplasm. The formulation exhibited comparable cytotoxicity in MDA-MB-231 cells (IC50 = 2.73 µM, 36 h), and inhibited the migration of cancer cells significantly compared to the free drug and unloaded PDA NPs. Furthermore, the unloaded NPs exhibited excellent in vivo compatibility. The study establishes a rigorously optimized protocol for the synthesis of Nic loaded PDA NPs. The biocompatibility, anti-migratory efficacy, and the in vivo non-toxic nature of PDA has been well demonstrated.
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Nanopartículas , Niclosamida , Humanos , Concentração de Íons de Hidrogênio , Indóis , Masculino , PolímerosRESUMO
The epithelial cell adhesion molecule (EpCAM) is a transmembrane cell adhesion glycoprotein, which primarily contributes to stemness, proliferation, and metastasis properties of tumor cells. Regulated intramembrane proteolysis by ADAM proteases and γ-secretase cleaves EpCAM into an â¼27 kDa soluble extracellular and an â¼4 kDa cytoplasmic domain (EpICD). After the EpICD fragment is released inside the cell, the formation of a nuclear signaling complex with the FHL2 molecule is critical for exerting its regulatory role. Trop-2, a homologous protein of EpCAM, undergoes phosphorylation in its cytoplasmic domain (Trop-IC). The phosphorylation of Trop-2 is reported to be crucial for its function. This led us to ask the fundamental question if EpCAM does undergo similar post-translational modification(PTM) like its homologous protein to carry out its diverse biological function. Here, we identify a putative phosphorylation site at Tyr297 located in the cytoplasmic domain of EpCAM. Molecular dynamic simulation (MDS) of 90 ns was carried out to understand the biological/functional relevance of the putative phosphorylation. It was observed that this phosphorylation stabilizes the α-helical structure of the EpICD. Though Tyr297 does not affect the γ-secretase mediated cleavage of EpCAM, it affects the binding of EpICD to FHL2. Docking analysis revealed that phosphorylation mediated structural stability of EpICD positively impacts its binding affinity with FHL2, which was further validated using 100 ns MDS. Phosphorylated EpICD forms higher numbers of hydrogen bonds, salt bridges, and other non-bonded interactions with FHL2, leading to enhanced interactions. This in silico study reveals a potential PTM in the EpICD, providing the basis for future research in understanding the mechanism behind the diverse biological function of EpCAM.
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Photothermal therapy (PTT), a simple and minimally invasive procedure, is an attractive option for cancer therapy. To date, inorganic agents have been widely employed as photothermal agents; however, organic molecules may provide a solution to rapid metabolic/in vivo clearance. Herein, we prepared lipid (S 75)-stabilized meso-tritolyl-BF2-oxasmaragdyrin nanoparticles (TBSNPs) using thin-film hydration and homogenization. Assessment of the physicochemical properties of the TBSNPs reveals the formation of particles of size <12 nm stabilized within the lipid matrix. The TBSNPs exhibit near infrared fluorescence (NIRF) being accompanied by an increase in non-radiative decay, leading to excellent photothermal properties. In vitro studies demonstrate excellent biocompatibility, hemocompatibility, cellular internalization, and photothermal efficacy (p = 0.0004). Extensive in vivo assessment of TBSNPs also highlights the non-toxic nature of the material and passive tumor homing. The strong NIRF exhibited by the material is exploited for whole-body imaging in the rodent model. The novel material also shows excellent photothermal efficacy (p = 0.0002) in a 4T1 xenograft mice model. The organic nature of the material coupled with its small size and strong NIRF provides an advantage for bio-elimination and potential clinical image-guided therapy over the inorganic counterparts.
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Materiais Biocompatíveis/química , Nanopartículas/química , Pirróis/química , Animais , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/química , Camundongos , Camundongos Nus , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Tamanho da Partícula , Terapia Fototérmica , Espectroscopia de Luz Próxima ao Infravermelho , Nanomedicina Teranóstica , Distribuição Tecidual , Transplante HomólogoRESUMO
Human sodium iodide symporter (NIS) gene mediated radio-ablation is a successful procedure in thyroid cancer clinics. In recent years, natural expression of NIS is reported in breast cancer (BC) cases but is yet to make its mark as a therapeutic procedure in BC clinics. A pre-exposure to histone deacetylase (HDAC) inhibitors to amplify endogenous NIS expression was attempted, but achieving cancer tissue-specific enhancement of NIS in patients is an important challenge to win. Here, for the first time, we show that a benzamide class of HDACi (bHDACi) can significantly induce NIS gene expression and function (p < 0.05) in BC cells with minimal off-target effects. Transcription factor (TF) profiler and promoter binding array reveals 22 TFs differentially activated by CI-994, of which FOXA1 is identified as a unique and positive regulator of NIS. Clonogenic assay shows reduced survival with bHDACi + 131I combination treatment. Further, AR-42 and MS-275 treatment shows enhanced NIS expression in an orthotopic breast tumor model. Combining bHDACi with 1 mCi 131I shows 40% drop in signal (p < 0.05), indicating enhanced radio-ablation effect. Cerenkov imaging revealed higher accumulation of 131I in MS-275-treated tumors. Thus, bHDACi-mediated selective enhancement ensuring minimal off-target effect is a step further toward using NIS as a therapeutic target for BC.
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Activation of STAT3 via Y705-phosphorylation is well documented across multiple cancer types and thus forms the basis of canonical pathway to judge STAT3 activation. Recently, important roles of two other post translational modification (PTM) sites, i.e. S727-phosphorylation and K685-acetylation, leading to STAT3 activation are reported. However, their critical mode of function in controlling STAT3 dimerization and signaling, independent of canonical activation remains elusive. Therefore, to understand the functional relevance of each STAT3 PTMs in breast cancer (BC), cell models are developed by stable overexpression of PTM-site specific point mutants, i.e. Y705F, S727A or K685R, in a 3'UTR-STAT3 knockdown BC cell background. Results using this model system reveal novel findings showing that phosphorylation at S727 can lead to STAT3 activation independent of phosphoY705. We also demonstrate that loss of pS727 or K685ac significantly affects functional phenotypes such as cell survival and proliferation as well as downstream transcriptional activity (Twist 1, Socs3, c-Myc, Bcl-1 and Mcl-1) of STAT3. Thereafter, by utilizing a BRET biosensor for measuring STAT3 phosphorylation in live cells, a crucial role of pS727 in dictating STAT3 activation and homodimerization formation is uncovered. Further by performing retrospective IHC analysis of total and phospho-forms of STAT3 in a cohort of 76 triple negative breast cancer (TNBC) patient samples, a significant dominant expression of phosphoS727 over phosphoY705 PTM (p < 0.001) is found in STAT3 positive cases. We also focus on validating known STAT3 inhibitor molecules for their action against both pY705 and pS727 activation. This study for the first time demonstrates that an anti-helminth drug compound, Niclosamide, is capable of inactivating both phospho-PTM sites on STAT3 and exhibits excellent anticancer efficacy in preclinical TNBC tumour model.