We first confirmed podophyllotoxin as the principal bioactive constituent in Qinghai-derived Sinopodophyllum hexandrum. Then, using the purified compound, we evaluated its inhibitory effects and mechanism in MFC gastric cancer cells and a syngeneic subcutaneous tumor model. Podophyllotoxin from Qinghai Sinopodophyllum hexandrum was identified by FT-UHR LC-MS. Its cytotoxicity against MFC gastric cancer cells was measured by CCK-8 assay. Cell proliferation, migration, invasion, and apoptosis were determined by colony formation, wound healing, Transwell, and flow cytometry assays, respectively. A syngeneic subcutaneous tumor model was established in 32 BALB/c mice for in vivo evaluation. Transcriptome sequencing was performed to profile gene expression in treated MFC cells, and Reverse transcription quantitative real-time PCR (RT-qPCR) verified key genes in MFC cells and tumor tissues. Podophyllotoxin significantly suppressed MFC cell proliferation, colony formation, migration, and invasion (P < 0.05), and induced early apoptosis (P < 0.05). In vivo, it inhibited tumor growth in a dose-dependent manner (P < 0.05). Transcriptome analysis showed that podophyllotoxin affected the DNA damage response pathway. RT-qPCR verified altered expression of DNA damage-related genes including Id3, Id4, Polq, Ddias, Gadd45a, Cip2a, Recql4, Rad54b, Mastl, and Fanci. Western blot confirmed downregulation of ID4 protein. Podophyllotoxin inhibits the growth of MFC gastric cancer cells and syngeneic subcutaneous tumors, probably by modulating the expression of DNA damage response-related genes. ID4 downregulation may serve as a potential target in this experimental model.
Three-dimensional (3D) cell culture models, including tumor spheroids and organoids, more accurately recapitulate the structural and physiological complexity of tissues than conventional monolayer cultures, making them valuable platforms for cancer research and drug screening. Volume is a biologically relevant parameter for monitoring growth dynamics and treatment response; however, its quantification often relies on manual image analysis, which is labor-intensive, subjective, and prone to inter-operator variability. Here, we introduce SpheroQuaNNt, a web-based application for automated spheroid segmentation and volume estimation using a U-Net Convolutional Neural Network (CNN). SpheroQuaNNt enables analysis of heterogeneous morphologies through image-based segmentation and provides a web-based interface for volume measurement. The model was trained and validated using 615 microscopy images representing diverse 3D culture systems, including monoculture and hybrid spheroids generated from human and canine tumor cell lines and human embryonic kidney cells, as well as tumor organoids derived from human melanoma lesions. After 30 training epochs, the model achieved 99.35
The role of cyclin-dependent kinase inhibitor 1B (CDKN1B) in non-alcoholic fatty liver disease (NAFLD) and its underlying mechanism remain unclear. In the present study, NAFLD models were constructed in high-fat diet (HFD)-fed mice and free fatty acid (FFA)-treated HepG2 cells. The expression of CDKN1B, markers of liver injury, inflammation, and mitophagy (PINK1, Parkin, Beclin1, and the LC3II/I ratio) were assessed. CDKN1B overexpression and knockdown were performed in HepG2 cells to investigate the functional roles of CDKN1B. The phase separation potential of CDKN1B was predicted bioinformatically and validated via in vitro droplet assays, immunofluorescence, and fluorescence recovery after photobleaching (FRAP). To determine whether the protective effects of CDKN1B depend on its phase separation property, the phase separation inhibitor 1,6-hexanediol (1,6-HD) was applied at graded concentrations. The results indicated that CDKN1B was downregulated in both in vivo and in vitro NAFLD models, accompanied by lipid accumulation, inflammation, mitochondrial dysfunction, and impaired mitophagy. CDKN1B overexpression effectively reversed these pathologies, whereas CDKN1B knockdown exacerbated them. Moreover, CDKN1B contains an intrinsically disordered region and undergoes liquid-liquid phase separation (LLPS) both in vitro and in HepG2 cells. Critically, 1,6-HD abolished the protective effects of CDKN1B overexpression on mitophagy, mitochondrial integrity, lipid metabolism, and inflammation in a concentration-dependent manner. Collectively, CDKN1B alleviates NAFLD by promoting mitophagy, and this protective function is dependent on its ability to undergo LLPS. This study unveils a novel phase-separation-dependent mechanism of CDKN1B in regulating mitochondrial function and mitophagy, presenting CDKN1B condensates as a potential new target for NAFLD therapy.
Epilepsy is a chronic neurological disorder, more common in children and the elderly. Although treatments have advanced, significant challenges remain. This study focuses on its molecular mechanisms to provide new insights and strategies for its treatment. Gene expression omnibus (GEO) database analysis, enrichment analysis, and the establishment of protein-protein interaction (PPI) networks were conducted for gene screening. HT-22 cells were treated with glutamate (Glu) to establish an in vitro epilepsy model. Gene knockdown and overexpression were achieved through cell transfection. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while gene expression at the protein and messenger RNA (mRNA) levels was detected by western blot (WB) and real-time quantitative polymerase chain reaction (RT-qPCR). Flow cytometry was used to analyze apoptosis and mitochondrial membrane potential. Lactate dehydrogenase (LDH), reactive oxygen species (ROS), and Fe2+ levels were measured using commercial kits. Kainic acid (KA) was administered intraperitoneally to mice to establish an epilepsy animal model, which was used to verify the role of the gene in epilepsy development. Transcription factor binding sites in target gene promoters were predicted using the JASPAR database and validated by chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. Tissue inhibitor of metalloproteinases 1 (Timp1) was highly expressed in tissue samples from the epilepsy mouse models of GSE88992 and GSE73878, as well as in the Glu-induced HT-22 cell model. In these models, Timp1 knockdown increased cell viability, reduced apoptosis, decreased LDH, promoted mitochondrial membrane potential, and lowered ROS, Fe2+, and acyl-CoA synthetase long-chain family member 4 (Acsl4) levels. In KA-induced animal models, Timp1 knockdown reduced seizure score, cleaved caspase-3 (C-caspase3)/total caspase-3, and Acsl4 expression. cAMP response element-binding protein 1 (Creb1) transcriptionally activated Timp1. In the Glu-induced HT-22 cell model, Creb1 knockdown decreased Timp1 protein expression, increased cell viability and mitochondrial membrane potential, and reduced apoptosis, LDH, ROS, Fe2+, and Acsl4 levels, while Timp1 overexpression partially reversed these effects. Taken together, Creb1 promotes the progression of epilepsy by activating Timp1, providing a new perspective and potential targets for epilepsy treatment.
