RESUMEN
BACKGROUND: Tiliroside (TIL) is a flavonoid compound that exists in a variety of edible plants. These dietary plants are widely used as food and medicine to treat various diseases. However, the effect of TIL on pancreatic cancer (PC) and its underlying mechanisms are unclear. PURPOSE: This study aims to reveal the anti-PC effect of TIL and clarify its mechanism. METHODS: The inhibitory effects of TIL on PC growth were studied both in vitro and in vivo. Flow cytometry, transmission electron microscopy, immunofluorescence, biochemical analyses, RT-qPCR, genetic ablation, and western blotting were employed to evaluate ferroptosis, autophagy, and iron regulation. Additionally, RNA sequencing (RNA-seq), biomolecular layer interferometry (BLI), and molecular simulation analysis were combined to identify TIL molecular targets. The clinicopathological significance of Calpain-2 (CAPN2) was determined through immunohistochemistry (IHC) on a PC tissue microarray. RESULTS: Herein, we showed that TIL was an effective anti-PC drug. CAPN2 was involved in the TIL - induced elevation of the labile iron pool (LIP) in PC cells. TIL directly bound to and inhibited CAPN2 activity, resulting in AKT deactivation and decreased expression of glucose transporters (GLUT1 and GLUT3) in PC cells. Consequently, TIL impaired ATP and NADPH generation, inducing autophagy and ROS production. The accumulation of TIL-induced ROS combined with LIP iron causes the Fenton reaction, leading to lipid peroxidation. Meanwhile, TIL-induced reduction of free iron ions promoted autophagic degradation of ferritin to regulate cellular iron homeostasis, which further exacerbated the death of PC cells by ferroptosis. As an extension of these in vitro findings, our murine xenograft study showed that TIL inhibited the growth of PANC-1 cells. Additionally, we showed that CAPN2 expression levels were related to clinical prognoses in PC patients. CONCLUSION: We identify TIL as a potent bioactive inhibitor of CAPN2 and an anti-PC candidate of natural origin. These findings also highlight CAPN2 as a potential target for PC treatment.
Asunto(s)
Ferroptosis , Neoplasias Pancreáticas , Humanos , Animales , Ratones , Calpaína/genética , Calpaína/farmacología , Especies Reactivas de Oxígeno/metabolismo , Flavonoides/farmacología , Neoplasias Pancreáticas/patología , Hierro/metabolismo , HomeostasisRESUMEN
BACKGROUND: Combination therapy with other antineoplastic agent is a favorable approach for targeting the molecules involved in sorafenib resistance. PURPOSE: In the present study, we determined whether tiliroside, a natural flavonoid glycoside isolated from oriental paperbush flower, could improve the sensitivity of hepatocellular carcinoma (HCC) cells to sorafenib. Furthermore, we investigated the mechanisms and identified the potential drug targets of tiliroside. METHODS: Synergy was performed using CalcuSyn. Transcriptomic studies were adopted to investigate whether tiliroside could induce ferroptosis and inhibit the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in HCC cells. Ferroptosis was analyzed using western blotting, flow cytometry, and transmission electron microscopy. Immunofluorescence, co-immunoprecipitation, and Nrf2 knockdown or overexpression were performed to confirm the involvement of Nrf2 in tiliroside-induced ferroptosis. Additionally, molecular docking and biolayer interferometry-based measurements were used to confirm the direct target of tiliroside. Finally, subcutaneous xenograft and orthotopic xenograft tumors in nude mice were used to assess the effects of tiliroside in vivo. RESULTS: Tiliroside significantly enhanced the anti-HCC activity of sorafenib without any discernible side effects. Moreover, the combination of tiliroside and sorafenib induced synergistic effects against HCC in vitro. The inhibitory effects of tiliroside on HCC were antagonized by N-acetylcysteine and the ferroptosis inhibitor liproxstatin-1. Studies on the mechanism of action revealed that tiliroside could directly bind to TANK-binding kinase 1 (TBK1) and inhibit its enzymatic activity. Inhibition of TBK1 by tiliroside decreased the phosphorylation of serine 349 on sequestosome-1 (p62) and the affinity of p62 for kelch like ECH-associated protein 1 (Keap1) and promoted Keap1-mediated Nrf2 ubiquitination and degradation. The downstream target proteins of Nrf2, including glutathione peroxidase 4, ferritin heavy chain 1, and glucose-6-phosphate dehydrogenase, demonstrated similar results to that of Nrf2 protein, inducing ferroptosis in tiliroside-treated HCC cells. We extended these findings in vivo and found that tiliroside inhibited the growth of HepG2 tumors in both subcutaneous xenograft and orthotopic xenograft tumor models of HCC. CONCLUSION: Our findings imply that tiliroside is a potent TBK1 inhibitor and a candidate natural anti-cancer product that could function as a sensitizer of sorafenib in HCC treatment by targeting TBK1 to induce ferroptosis.
Asunto(s)
Carcinoma Hepatocelular , Ferroptosis , Neoplasias Hepáticas , Animales , Ratones , Humanos , Carcinoma Hepatocelular/patología , Sorafenib/farmacología , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Hepáticas/patología , Ratones Desnudos , Factor 2 Relacionado con NF-E2/metabolismo , Simulación del Acoplamiento Molecular , Flavonoides/uso terapéutico , Línea Celular Tumoral , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Astragalin (AG) is a biologically active flavonoid compound that can be extracted from a number of medicinal plants. However, the effects of AG on osteoblastic differentiation in mouse MC3T3-E1 cells and on bone formation in vivo have not been studied fully. In this study, we found that the activities of alkaline phosphatase (ALP) and mineralized nodules in MC3T3-E1 cells were both significantly increased after treatment with AG (5, 10, and 20 µM). Meanwhile, the mRNA and protein levels of osteoblastic marker genes in MC3T3-E1 cells after AG treatment were markedly increased compared with a control group. In addition, the levels of BMP-2, p-Smad1/5/9, and Runx2 were significantly elevated in AG-treated MC3T3-E1 cells. Moreover, we found that the protein levels of Erk1/2, p-Erk1/2, p38, p-p38, and p-JNK were also significantly increased in AG-treated MC3T3-E1 cells compared to those in the control group. Finally, in vivo experiments demonstrated that AG significantly promoted bone formation in an ovariectomized (OVX)-induced osteoporotic mouse model. This was evidenced by significant increases in the values of osteoblast-related parameters (BFR/BS, MAR, Ob.S/BS, and Ob.N/B.Pm) and bone histomorphometric parameters (BMD, BV/TV, Tb.Th, and Tb.N.) in OVX mice after AG treatment (5, 10, and 20 mg/kg). Collectively, these results demonstrated that AG may promote osteoblastic differentiation in MC3T3-E1 cells via the activation of the BMP and MAPK pathways and promote bone formation in vivo. These novel findings indicated that AG may be a useful bone anabolic agent for the prevention and treatment of osteoporosis.