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1.
STAR Protoc ; 5(2): 103058, 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38748881

RESUMO

Three-dimensional (3D) models play an increasingly important role in preclinical drug testing as they faithfully mimic interactions between cancer cells and the tumor microenvironment (TME). Here, we present a protocol for generating scaffold-free 3D multicomponent human melanoma spheroids. We describe steps for characterizing models using live-cell imaging and histology, followed by drug testing and assessment of cell death through various techniques such as imaging, luminescence-based assays, and flow cytometry. Finally, we demonstrate the models' adaptability for co-cultures with immune cells.


Assuntos
Melanoma , Esferoides Celulares , Humanos , Esferoides Celulares/patologia , Esferoides Celulares/metabolismo , Melanoma/patologia , Melanoma/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Microambiente Tumoral , Técnicas de Cocultura/métodos , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Técnicas de Cultura de Células/métodos
2.
Cell Death Dis ; 14(12): 831, 2023 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-38097548

RESUMO

Malignant melanoma (MM) is known to be intrinsically chemoresistant, even though only ~20% of MM carry mutations of the tumor suppressor p53. Despite improvement of systemic therapy the mortality rate of patients suffering from metastatic MM is still ~70%, highlighting the need for alternative treatment options or for the re-establishment of conventional therapeutic approaches, including chemotherapy. Screening the p53 mutation status in a cohort of 19 patient-derived melanoma samples, we identified one rarely described missense mutation of p53 leading to E285K amino acid exchange (mutp53(E285K)). Employing structural and computational analysis we revealed a major role of E285 residue in maintaining stable conformation of wild-type p53 (wtp53). E285K mutation was predicted to cause interruption of a salt-bridge network affecting the conformation of the C-terminal helix of the DNA-binding domain (DBD) thereby preventing DNA interaction. In this context, a cluster of frequently mutated amino acid residues in cancer was identified to putatively lead to similar structural effects as E285K substitution (E285 cluster). Functional analysis, including knockdown of endogenous p53 and reconstitution with diverse p53 missense mutants confirmed mutp53(E285K) to have lost transcriptional activity, to be localized in the cytosol of cancer cells, by both means conferring chemoresistance. Re-sensitization to cisplatin-induced cell death was achieved using clinically approved compounds aiming to restore p53 wild-type function (PRIMA1-Met), or inhibition of AKT-driven MAPK survival pathways (afuresertib), in both cases being partially due to ferroptosis induction. Consequently, active ferroptosis induction using the GPX4 inhibitor RSL3 proved superior in tumorselectively fighting MM cells. Due to high prevalence of the E285-cluster mutations in MM as well as in a variety of other tumor types, we conclude this cluster to serve an important function in tumor development and therapy and suggest new implications for ferroptosis induction in therapeutic applications fighting MM in particular and cancer in general.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Melanoma , Neoplasias Cutâneas , Proteína Supressora de Tumor p53 , Humanos , Aminoácidos , Linhagem Celular Tumoral , Citosol/metabolismo , DNA , Resistencia a Medicamentos Antineoplásicos/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Mutação , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Proteína Supressora de Tumor p53/metabolismo
3.
Cancer Gene Ther ; 30(10): 1330-1345, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37420093

RESUMO

Therapy Induced Senescence (TIS) leads to sustained growth arrest of cancer cells. The associated cytostasis has been shown to be reversible and cells escaping senescence further enhance the aggressiveness of cancers. Chemicals specifically targeting senescent cells, so-called senolytics, constitute a promising avenue for improved cancer treatment in combination with targeted therapies. Understanding how cancer cells evade senescence is needed to optimise the clinical benefits of this therapeutic approach. Here we characterised the response of three different NRAS mutant melanoma cell lines to a combination of CDK4/6 and MEK inhibitors over 33 days. Transcriptomic data show that all cell lines trigger a senescence programme coupled with strong induction of interferons. Kinome profiling revealed the activation of Receptor Tyrosine Kinases (RTKs) and enriched downstream signaling of neurotrophin, ErbB and insulin pathways. Characterisation of the miRNA interactome associates miR-211-5p with resistant phenotypes. Finally, iCell-based integration of bulk and single-cell RNA-seq data identifies biological processes perturbed during senescence and predicts 90 new genes involved in its escape. Overall, our data associate insulin signaling with persistence of a senescent phenotype and suggest a new role for interferon gamma in senescence escape through the induction of EMT and the activation of ERK5 signaling.


Assuntos
Insulinas , Melanoma , Humanos , Multiômica , Linhagem Celular Tumoral , Melanoma/tratamento farmacológico , Melanoma/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Insulinas/uso terapêutico , Senescência Celular/genética , Proteínas de Membrana/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/uso terapêutico
4.
ACS Chem Neurosci ; 12(7): 1049-1060, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33687205

RESUMO

Alzheimer's disease (AD) is characterized by the presence of ß-amyloid plaques (Aß) and neurofibrillary tangles (NFTs) in the brain. The prevalence of the disease is increasing and is expected to reach 141 million cases by 2050. Despite the risk factors associated with the disease, there is no known causative agent for AD. Clinical trials with many drugs have failed over the years, and no therapeutic has been approved for AD. There is increasing evidence that pathogens are found in the brains of AD patients and controls, such as human herpes simplex virus-1 (HSV-1). Given the lack of a human model, the route for pathogen entry into the brain remains open for scrutiny and may include entry via a disturbed blood-brain barrier or the olfactory nasal route. Many factors can contribute to the pathogenicity of HSV-1, such as the ability of HSV-1 to remain latent, tau protein phosphorylation, increased accumulation of Aß invivo and in vitro, and repeated cycle of reactivation if immunocompromised. Intriguingly, valacyclovir, a widely used drug for the treatment of HSV-1 and HSV-2 infection, has shown patient improvement in cognition compared to controls in AD clinical studies. We discuss the potential role of HSV-1 in AD pathogenesis and argue for further studies to investigate this relationship.


Assuntos
Doença de Alzheimer , Herpes Simples , Herpesvirus Humano 1 , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides , Humanos , Emaranhados Neurofibrilares , Placa Amiloide
5.
Vet Pathol ; 58(2): 325-331, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33208018

RESUMO

Using immunohistochemistry, 170 canine mammary carcinomas were evaluated for p53, ER (estrogen receptor), and Ki67. Of the 170 tumors, 89 were grade I (52.3%), 36 were grade II (21.2%), and 45 were grade III (26.4%). Eight cases (0.5%) were positive for p53 and 69/170 cases (40.5%) were positive for ER. Ki67 values were 24 ± 18% (mean ± SD). Using a cutoff value of 33.3% Ki67-positive neoplastic nuclei, 38/159 (23.8%) were classified as high proliferative and 121/159 (76.2%) as low proliferative. p53-positive cases had significantly higher Ki67 expression and higher histological grade. ER expression was not correlated with p53 expression but was significantly related to low Ki67 values and low histological grade. Moreover, ER-positive cases had significantly longer survival compared to ER-negative tumors, and ER expression had better correlation with tumor-related survival than histological grade. In summary, p53 accumulated in a small subset of canine mammary tumors and was associated with higher proliferative activity and higher histological grade. ER expression was confirmed as a differentiation marker associated with more favorable prognosis and biological behavior. The combined use of these 3 markers could be used in addition to histological grade to predict the biological behavior of canine mammary carcinomas.


Assuntos
Neoplasias da Mama , Carcinoma , Doenças do Cão , Animais , Biomarcadores Tumorais , Neoplasias da Mama/veterinária , Carcinoma/veterinária , Doenças do Cão/diagnóstico , Cães , Feminino , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Prognóstico , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteína Supressora de Tumor p53/genética
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