RESUMEN
Drug resistance is a key factor underlying the failure of tumor chemotherapy. It enhances the stemlike cell properties of cancer cells, tumor metastasis and relapse. Luteolin is a natural flavonoid with strong antitumor effects. However, the mechanism(s) by which luteolin protects against paclitaxel (PTX)resistant cancer cell remains to be elucidated. The inhibitory effect of luteolin on the proliferation of EC1/PTX and EC1 cells was detected by cell counting kit8 assay. Colony formation and flow cytometry assays were used to assess clonogenic capacity, cell cycle and apoptosis. Wound healing and Transwell invasion tests were used to investigate the effects of luteolin on the migration and invasion of EC1/PTX cells. Western blotting was used to detect the protein levels of EMTrelated proteins and stem cell markers after sphere formation. Parental cells and drugresistant cells were screened by highthroughput sequencing to detect the differential expression of RNA and differential genes. ELISA and western blotting were used to verify the screened PI3K/Akt signaling pathway, key proteins of which were explored by molecular docking. Hematoxylin and eosin staining and TUNEL staining were used to observe tumor xenografts on morphology and apoptosis in nude mice. The present study found that luteolin inhibited tumor resistance (inhibited proliferation, induced cell cycle arrest and apoptosis and hindered migration invasion, EMT and stem cell spherification) in vitro in PTXresistant esophageal squamous cell carcinoma (ESCC) cells. In addition, luteolin enhanced drug sensitivity and promoted the apoptosis of drugresistant ESCC cells in combination with PTX. Mechanistically, luteolin may inhibit the PI3K/AKT signaling pathway by binding to the active sites of focal adhesion kinase (FAK), Src and AKT. Notably, luteolin lowered the tumorigenic potential of PTXresistant ESCC cells but did not show significant toxicity in vivo. Luteolin enhanced drug chemosensitivity by downregulating the FAK/PI3K/AKT pathway in PTXresistant ESCC and could be a promising agent for the treatment of PTXresistant ESCC cancers.
Asunto(s)
Resistencia a Antineoplásicos , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Luteolina , Paclitaxel , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Luteolina/farmacología , Paclitaxel/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Línea Celular Tumoral , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/metabolismo , Carcinoma de Células Escamosas de Esófago/patología , Carcinoma de Células Escamosas de Esófago/genética , Transducción de Señal/efectos de los fármacos , Ratones , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Ratones Desnudos , Movimiento Celular/efectos de los fármacos , Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 1 de Adhesión Focal/genética , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Ratones Endogámicos BALB C , Ensayos Antitumor por Modelo de Xenoinjerto , Antineoplásicos Fitogénicos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , MasculinoRESUMEN
Macroautophagy/autophagy is a catabolic process crucial for degrading cytosolic components and damaged organelles to maintain cellular homeostasis, enabling cells to survive in extreme extracellular environments. ENAH/MENA, a member of the Ena/VASP protein family, functions as a highly efficient actin elongation factor. In this study, our objective was to explore the role of ENAH in the autophagy process. Initially, we demonstrated that depleting ENAH in cancer cells inhibits autophagosome formation. Subsequently, we observed ENAH's colocalization with MAP1LC3/LC3 during tumor cell starvation, dependent on actin cytoskeleton polymerization and the interaction between ENAH and BECN1 (beclin 1). Additionally, mammalian ATG9A formed a ring-like structure around ENAH-LC3 puncta during starvation, relying on actin cytoskeleton polymerization. Furthermore, ENAH's EVH1 and EVH2 domains were found to be indispensable for its colocalization with LC3 and BECN1, while the PRD domain played a crucial role in the formation of the ATG9A ring. Finally, our study revealed ENAH-led actin comet tails in autophagosome trafficking. In conclusion, our findings provide initial insights into the regulatory role of the mammalian actin elongation factor ENAH in autophagy.Abbreviations: 3-MA 3-methyladenine; ABPs actin-binding proteins; ATG autophagy related; ATG9A autophagy related 9A; Baf A1 bafilomycin A1; CM complete medium; CytERM endoplasmic reticulum signal-anchor membrane protein; Cyto D cytochalasin D; EBSS Earl's balanced salt solution; ENAH/MENA ENAH actin regulator; EVH1 Ena/VASP homology 1 domain; EVH2 Ena/VASP homology 2 domain; GAPDH glyceraldehyde-3-phosphate dehydrogenase; Lat B latrunculin B; LC3-I unlipidated form of LC3; LC3-II phosphatidylethanolamine-conjugated form of LC3; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; mEGFP monomeric enhanced green fluorescent protein; mTagBFP2 monomeric Tag blue fluorescent protein 2; OSER organized smooth endoplasmic reticulum; PRD proline-rich domain; PtdIns3K class III phosphatidylinositol 3-kinase; WM wortmannin.