RESUMEN
Anlotinib is used for the treatment of advanced non-small cell lung cancer; however, the emergence of drug resistance limits its clinical application. ß-sitosterol may also be used to treat lung cancer, but there have been no studies evaluating ß-sitosterol against anlotinib-resistant lung cancer. The purpose of this study was to determine the mechanism by which ß-sitosterol enhances the sensitivity of lung cancer cells to anlotinib. A549 cells were treated with different concentrations of anlotinib to generate anlotinib-resistant cells (A549/anlotinib cells). miR-181a-3p mimics were transfected into A549/anlotinib cells. A549 and A549/anlotinib cells were treated with ß-sitosterol at various concentrations. The Cell Counting Kit-8 (CCK-8) assay was used to measure cell proliferation. Apoptosis was assessed by flow cytometry. Real-time quantitative PCR was used to measure the expression of miR-181a-3p. The interaction of miR-181a-3p with the H/ACA ribonucleoprotein assembly factor (SHQ1) was predicted using the miRDB and TargetScan Human databases and verified with a luciferase reporter assay. The expression of SHQ1, activating transcription factor 6 (ATF6), and glucose-regulated protein 78 (GRP78) were measured by western blot analysis. ß-Sitosterol effectively suppressed A549/anlotinib cell proliferation and promoted apoptosis. SHQ1 is a downstream target of miR-181a-3p. The expression of miR-181a-3p was inhibited; however, SHQ1 expression was increased by ß-sitosterol treatment of A549/anlotinib cells. The inhibition of SHQ1, ATF6, and GRP78 protein expression by ß-sitosterol in A549/anlotinib cells was rescued by increased miR-181a-3p. ß-Sitosterol markedly promotes anlotinib-resistant A549 cell apoptosis and inhibits cell proliferation by activating SHQ1/UPR signaling through miR-181a-3p inhibition.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Indoles , Neoplasias Pulmonares , MicroARNs , Quinolinas , Sitoesteroles , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Chaperón BiP del Retículo Endoplásmico , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , MicroARNs/efectos de los fármacos , MicroARNs/genética , Resistencia a Antineoplásicos/efectos de los fármacosRESUMEN
This study aims to explore the effects of Shenqi Dihuang Decoction on high-glucose induced ferroptosis and the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/glutathione peroxidase 4(GPX4) axis in human renal tubular epithelial cells(HK-2) and to clarify the underlying mechanism. The cell injury model was established by exposing HK-2 to high glucose, and the Shenqi Dihuang Decoction-medicated serum was prepared. The optimal concentration and intervention time of Shenqi Dihuang Decoction were determined. HK-2 were divided into normal, high glucose, and low-, medium-, and high-dose Shenqi Dihuang Decoction groups. After interventions, the cell proliferation rate in each group was determined and the cell morphology and mitochondrial ultrastructure were observed. Then, the levels of intracellular reactive oxygen species(ROS), ferrous ion(Fe~(2+)), glutathione(GSH), and malondialdehyde(MDA) and the protein levels of Nrf2, HO-1, GPX4, and xCT were measured. The optimal concentration and intervention time of Shenqi Dihuang Decoction-medicated serum were determined to be 10% and 24 h, respectively. Compared with the high glucose group, high-dose Shenqi Dihuang Decoction promoted the proliferation of HK-2. The cells in the low-, medium-, and high-dose Shenqi Dihuang Decoction groups presented tight arrangement, an increased cell count, improved morphology from a spindle-fiber shape to a cobblestone shape, and improved morphology and structure of mitochondrial membrane and cristae, compared with those in the high glucose group. Meanwhile, all the doses of Shenqi Dihuang Decoction inhibited ROS elevation to mitigate the peroxidation damage, lowered the Fe~(2+) and MDA levels and elevated the GSH level to inhibit lipid peroxidation, and activated the antioxidant pathway to upregulate the protein levels of Nrf2, HO-1, xCT, and GPX4. In conclusion, Shenqi Dihuang Decoction-medicated serum can inhibit high-glucose induced ferroptosis of HK-2 in vitro, which involves the antioxidant effect and the activation of the Nrf2/HO-1/GPX4 pathway.