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1.
Br J Cancer ; 121(9): 758-767, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31551581

RESUMO

BACKGROUND: Protoporphyrin IX (PpIX) gets accumulated preferentially in 5-aminolevulinic acid (5-ALA)-treated cancer cells. Photodynamic therapy (PDT) utilises the accumulated PpIX to trigger cell death by light-induced generation of reactive oxygen species (ROS). We previously demonstrated that oncogenic Ras/MEK decreases PpIX accumulation in cancer cells. Here, we investigated whether combined therapy with a MEK inhibitor would improve 5-ALA-PDT efficacy. METHODS: Cancer cells and mice models of cancer were treated with 5-ALA-PDT, MEK inhibitor or both MEK inhibitor and 5-ALA-PDT, and treatment efficacies were evaluated. RESULTS: Ras/MEK negatively regulates the cellular sensitivity to 5-ALA-PDT as cancer cells pre-treated with a MEK inhibitor were killed more efficiently by 5-ALA-PDT. MEK inhibition promoted 5-ALA-PDT-induced ROS generation and programmed cell death. Furthermore, the combination of 5-ALA-PDT and a systemic MEK inhibitor significantly suppressed tumour growth compared with either monotherapy in mouse models of cancer. Remarkably, 44% of mice bearing human colon tumours showed a complete response with the combined treatment. CONCLUSION: We demonstrate a novel strategy to promote 5-ALA-PDT efficacy by targeting a cell signalling pathway regulating its sensitivity. This preclinical study provides a strong basis for utilising MEK inhibitors, which are approved for treating cancers, to enhance 5-ALA-PDT efficacy in the clinic.


Assuntos
Ácidos Levulínicos/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Animais , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/metabolismo , Protoporfirinas/metabolismo , Distribuição Aleatória , Espécies Reativas de Oxigênio/metabolismo , Proteínas ras/metabolismo , Ácido Aminolevulínico
2.
Microbiol Spectr ; 9(1): e0043921, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34319169

RESUMO

Hepatitis C virus (HCV) can cause acute and chronic infection that is associated with considerable liver-related morbidity and mortality. In recent years, there has been a shift in the treatment paradigm with the discovery and approval of agents that target specific proteins vital for viral replication. We employed a cell culture-adapted strain of HCV and human hepatoma-derived cells lines to test the effects of our novel small-molecule compound (AO13) on HCV. Virus inhibition was tested by analyzing RNA replication, protein expression, and virus production in virus-infected cells treated with AO13. Treatment with AO13 inhibited virus spread in cell culture and showed a 100-fold reduction in the levels of infectious virus production. AO13 significantly reduced the level of viral RNA contained within cell culture fluids and reduced the cellular levels of HCV core protein, suggesting that the compound might act on a late step in the viral life cycle. Finally, we observed that AO13 did not affect the release of infectious virus from infected cells. Docking studies and molecular dynamics analyses suggested that AO13 might target the NS5B RNA polymerase, however, real-time RT-PCR analyses of cellular levels of HCV RNA showed only an ∼2-fold reduction in viral RNA levels in the presence of AO13. Taken together, this study revealed that AO13 showed consistent, but low-level antiviral effect against HCV, although the mechanism of action remains unclear. IMPORTANCE The discovery of curative antiviral drugs for a chronic disease such as HCV infection has encouraged drug discovery in the context of other viruses for which no curative drugs currently exist. Since we currently face a novel virus that has caused a pandemic, the need for new antiviral agents is more apparent than ever. We describe here a novel compound that shows a modest antiviral effect against HCV that could serve as a lead compound for future drug development against other important viruses such as SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Técnicas de Cultura de Células , Hepacivirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/uso terapêutico , Carcinoma Hepatocelular , Linhagem Celular , Hepacivirus/genética , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Hepatite C/virologia , Humanos , Estágios do Ciclo de Vida , Fígado , Neoplasias Hepáticas , Simulação de Acoplamento Molecular , RNA Viral , SARS-CoV-2 , Proteínas não Estruturais Virais , Liberação de Vírus/efeitos dos fármacos
3.
Theranostics ; 8(8): 2134-2146, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29721068

RESUMO

Protoporphyrin IX (PpIX) is an endogenous fluorescent molecule that selectively accumulates in cancer cells treated with the heme precursor 5-aminolevulinic acid (5-ALA). This cancer-specific accumulation of PpIX is used to distinguish tumor from normal tissues in fluorescence-guided surgery (FGS) and to destroy cancer cells by photodynamic therapy (PDT). In this study, we demonstrate that oncogenic Ras/mitogen-activated protein kinase kinase (MEK) pathway can modulate PpIX accumulation in cancer cells. Methods: To identify Ras downstream elements involved in PpIX accumulation, chemical inhibitors were used. To demonstrate the increase of PpIX accumulation by MEK inhibition, different human normal and cancer cell lines, BALB/c mice bearing mammary 4T1 tumors and athymic nude mice bearing human tumors were used. To identify the mechanisms of PpIX regulation by MEK, biochemical and molecular biological experiments were conducted. Results: Inhibition of one of the Ras downstream elements, MEK, promoted PpIX accumulation in cancer cells treated with 5-ALA, while inhibitors against other Ras downstream elements did not. Increased PpIX accumulation with MEK inhibition was observed in different types of human cancer cell lines, but not in normal cell lines. We identified two independent cellular mechanisms that underlie this effect in cancer cells. MEK inhibition reduced PpIX efflux from cancer cells by decreasing the expression level of ATP binding cassette subfamily B member 1 (ABCB1) transporter. In addition, the activity of ferrochelatase (FECH), the enzyme responsible for converting PpIX to heme, was reduced by MEK inhibition. Finally, we found that in vivo treatment with MEK inhibitors increased PpIX accumulation (2.2- to 2.4-fold) within mammary 4T1 tumors in BALB/c mice injected with 5-ALA without any change in normal organs. Similar results were also observed in a human tumor xenograft model. Conclusion: Our study demonstrates that inhibition of oncogenic Ras/MEK significantly enhances PpIX accumulation in vitro and in vivo in a cancer-specific manner. Thus, suppressing the Ras/MEK pathway may be a viable strategy to selectively intensify PpIX fluorescence in cancer cells and improve its clinical applications in FGS.


Assuntos
Genes ras , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Protoporfirinas/farmacologia , Transdução de Sinais , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Ácido Aminolevulínico/farmacologia , Animais , Linhagem Celular Tumoral , Feminino , Ferroquelatase/metabolismo , Fluorescência , Heme/metabolismo , Humanos , Imageamento Tridimensional , Camundongos , Camundongos Endogâmicos BALB C , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos
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