Cytotechnology最新文献

Metastasis is the process by which cancer cells are disseminated from their primary site to distant locations, causing complications in anti-cancer treatments and a high rate of mortality. Therefore, the discovery of novel drug candidates that can inhibit metastasis progression is crucial for improving anti-cancer therapeutic outcomes. Tauro ursodeoxycholic acid (TUDCA) is a bile acid with known cytoprotective and anti-apoptotic properties. In this study, we investigated the anti-metastatic activity of TUDCA in 12-O-tetradecanoylphorbol-13-acetate (PMA) stimulated human fibrosarcoma HT-1080 cells. TUDCA treatment decreased cell viability in a dose-dependent manner in HT-1080 cells. TUDCA displayed the antimetastatic effect by significantly inhibiting the cell invasion and migration in HT-1080 cells at 5, 10, and 20 µM (p < 0.05). Gelatin zymography revealed that TUDCA inhibited the MMP2 and MMP9 activities in HT-1080 cells in a concentration-dependent manner. Western blot analysis showed that TUDCA treatment inhibited the phosphorylation of MAPK pathway-related ERK, JNK, and p38, along with suppression of nuclear translocation of p-p65 and c-FOS, confirming that TUDCA could modulate MAPK and NF-κB pathways for anti-metastatic activity. Furthermore, gene reporter assay confirmed that TUDCA inhibited the transcriptional activity of NF-κB and AP1. These results suggest that TUDCA could exert a potent anti-metastatic activity in PMA-stimulated HT-1080 cells through modulation of MMPs, MAPKs signaling pathway, and NF-κB and AP1 transcription factors.

Acute lung injury (ALI) is a life-threatening critical condition with high mortality. Adipose mesenchymal stem cell (ADMSC)-derived exosomes hold great potential for clinical therapy and drug delivery. Given that glutathione S-transferase Pi 1 (GSTP1), a key enzyme in oxidative stress response, is highly expressed in lung tissue and has been implicated in mitigating inflammatory and oxidative damage. However, the research on GSTP1-mediated ADMSC-derived exosomes in ALI is still blank. ADMSCs were characterized using flow cytometry, oil red O, Alizarin Red S, and clone formation assays. Gene expression was detected by quantitative real-time PCR (qRT-PCR) and western blotting. Exosomes were isolated and characterized by nanoparticle tracking analysis (NTA) and transmission electron microscope (TEM), and their uptake was measured adopting the 3,3'-dioctadecyloxacarbocyanine perchlorate (DIO) probe. In vitro and in vivo ALI models were established exploiting lipopolysaccharide (LPS). Moreover, the cell viability, cytotoxicity, inflammatory response, cell apoptosis, oxidative stress markers, mitochondrial membrane potential, and mitochondrial DNA (mtDNA) and adenosine 5'-triphosphate (ATP) levels, as well as hematoxylin-eosin (H&E) staining were analyzed to evaluate the effects of GSTP1-modified exosomes on ALI. ADMSCs were positive for CD90 and CD73 and negative for CD34 and CD45. Exosomes were successfully isolated from ADMSCs, and GSTP1-modified exosomes exhibited enhanced uptake by human type II alveolar epithelial cells (HPAEpiCs). Furthermore, GSTP1-modified ADMSC exosomes attenuated LPS-induced inflammatory response, oxidative stress, mitochondrial dysfunction, and apoptosis in HPAEpiCs. GSTP1-modified exosomes derived from ADMSCs ameliorate ALI, suggesting a novel therapeutic target for this condition.

Graphical abstract: GSTP1 up-regulated ADMSC-derived exosomes inhibit inflammation, apoptosis, oxidative stress, and mitochondrial dysfunction in LPS-induced ALI of HPAEpiCs.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00863-y.

Background: Periodontitis (PD), driven by dysregulated inflammation and bone loss, remains a therapeutic challenge. AlkB homolog 5, RNA demethylase (ALKBH5), a regulator of N6-methyladenosine (m6A) modification, influences immune responses and osteogenesis, yet its role in PD is unexplored.

Methods: An in vitro PD model was established by treating human periodontal ligament stem cells (hPDLSCs) using lipopolysaccharide (LPS). Western blotting assay was performed to detect the protein expression of ALKBH5, toll-like receptor 9 (TLR9), inflammatory cytokines (IL-6, IL-1β, TNF-α), and osteogenic markers (RUNX2, OPN, OCN). The mRNA levels of ALKBH5, TLR9, IL-6, IL-1β and TNF-α were detected by quantitative real-time polymerase chain reaction. Osteogenic capacity was assessed by alizarin red S staining and alkaline phosphatase (ALP) activity. Methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation and actinomycin D assays were used to explore ALKBH5-TLR9 interactions and mRNA stability, respectively.

Results: Expression levels of both ALKBH5 and TLR9 were elevated in the gingival tissues of patients with PD as well as in hPDLSCs stimulated by LPS. LPS elevated IL-6, IL-1β, and TNF-α levels (mRNA/protein), but ALKBH5 knockdown reversed these effects. LPS suppressed mineralization, ALP activity, and the protein expression of RUNX2, OPN, and OCN, while ALKBH5 depletion restored these outcomes. Mechanistically, ALKBH5 silencing reduced TLR9 mRNA stability via m6A modification. Moreover, TLR9 overexpression relieved ALKBH5 knockdown-induced anti-inflammatory and pro-osteogenic effects in LPS-treated cells. Further, ALKBH5 silencing inactivated the MAPK pathway by regulating TLR9 in LPS-treated cells.

Conclusion: ALKBH5 stabilized TLR9 to activate the MAPK pathway, further amplifying inflammation and inhibiting osteogenic differentiation in LPS-induced hPDLSCs. Targeting the ALKBH5-TLR9 axis might alleviate periodontal inflammation and enhance regeneration, offering a novel therapeutic avenue for PD management.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00889-2.

Gastric cancer is a leading cause of cancer-related mortality worldwide, largely due to its late diagnosis, aggressive progression, and the adverse effects of current treatment options. There is a growing interest in identifying natural, less toxic alternatives, with plant-derived compounds offering promising therapeutic potential. Resveratrol, a polyphenol found in red grapes, has demonstrated notable anticancer effects by modulating several cellular pathways involved in tumor development. This study aimed to evaluate the anticancer effects of resveratrol and red grape extract on AGS human gastric adenocarcinoma cells, with a focus on cell viability, apoptosis, cell cycle dynamics, and gene expression. Red grape extract was prepared, and resveratrol solubility was systematically assessed in a panel of pharmaceutical excipients. AGS human gastric cancer cells and corresponding normal fibroblasts were cultured and subjected to controlled mechanical injury prior to treatment. Cytotoxicity of the extract and resveratrol was evaluated using the MTT assay to determine effects on cell viability. Apoptosis was quantified by flow cytometry using the Annexin V-FITC/propidium iodide (PI) assay. The expression levels of apoptosis-related genes were analyzed by quantitative real-time PCR (qRT-PCR). In addition, total antioxidant capacity (TAC) was measured to assess treatment-induced oxidative alterations. Using MTT assays, both resveratrol and red grape extract showed dose- and time-dependent inhibition of AGS cell proliferation, with resveratrol displaying a stronger effect (IC₅₀: 7.812 µg/mL) compared to red grape extract (IC₅₀: 31.25 µg/mL) at 48 and 72 h. In contrast, neither compound significantly affected the viability of normal HGF1-PI 1 fibroblast cells. Flow cytometry revealed that resveratrol induced higher levels of apoptosis (53.72%) in AGS cells than red grape extract (41.4%), with selective action favoring cancer cells over normal cells. Gene expression analysis via RT-qPCR demonstrated that resveratrol significantly upregulated pro-apoptotic gene MCC and anti-apoptotic ERBB2 gene, while downregulating anti-apoptotic genes CCND1, MICAL2, and PAK1 in AGS cells. No significant gene modulation occurred in normal cells. Additionally, resveratrol markedly decreased the total antioxidant capacity (TAC) in AGS cells, suggesting a pro-oxidant mechanism of action, while TAC in normal cells remained unchanged. In conclusion, resveratrol exhibits potent and selective anticancer effects on gastric cancer cells, including apoptosis induction, cell cycle arrest, and gene modulation, with minimal cytotoxicity toward normal fibroblasts. These findings underscore resveratrol's potential as a promising natural agent in gastric cancer therapy.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00884-7.

Multiple myeloma (MM) is a clonal plasma cell disorder and is the second most common hematologic malignancy worldwide. In recent years, ferroptosis has emerged as an important target for cancer therapy, including MM. Cordycepin (COR) is a nucleoside antibiotic that was first isolated from fungi, with potential anti-tumor properties. The current research elucidated the therapeutic mechanism against MM of COR by investigating its effects on ferroptosis. U266 or NCI-H929 cells were incubated with COR (1, 3, and 10 µM) for 24 h, respectively. In both U266 and NCI-H929 cells, dramatically declined cell viability, reduced migrated cell counts, increased ROS productions and MDA levels, and repressed SOD activities were observed following COR incubation. Furthermore, in COR-treated U266 or NCI-H929 cells, markedly increased Fe²⁺ levels, upregulated ACSL4, and downregulated GPX4 were induced by COR, accompanied by an inhibition of HIF-1α/SLC7A11 axis and an upregulation of CLEC2. To confirm the role of ferroptosis and CLEC2 in COR's anti-tumor function, U266 cells were treated by COR for 24 h, followed by incubation with Fer-1 or transfected with si-CLEC2. The decreased cell viability, reduced migrated cells, increased ACSL4 levels, downregulated GPX4, and inhibited HIF-1α/SLC7A11 axis in COR-treated U266 cells were remarkably reversed by Fer-1 or silencing CLEC2. In addition, inhibitory effects of COR on the growth of U266 xenograft model, as well as facilitating effects of COR on ferroptosis in U266 xenograft tumor tissues, were reversed by Fer-1 or silencing CLEC2. Collectively, COR inhibited MM progression by inducing ferroptosis through upregulating CLEC2.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00886-5.

Recurrent spontaneous abortion (RSA) is a major reproductive challenge with multifaceted causes, including genetic and immunological influences. Complement system-related genes (CRGs) have been implicated in RSA pathogenesis, yet their exact roles remain unclear. This study aims to identify related biomarkers for RSA and investigate the underlying mechanisms. Datasets GSE65099 and GSE26787 were obtained from the GEO database. The GSE65099 dataset was used to identify differentially expressed genes (DEGs), which were intersected with key module genes identified from WGCNA to obtain DE-CRGs related to RSA. Then, key CRGs were screened by combined PPI networks, machine learning, and external validation. A nomogram model was developed for the diagnosis of RSA with key CRG expression levels, followed by validation via ROC curve analysis. Transcription factor-key gene and chemical-gene networks were constructed to reveal the potential mechanisms of key CRGs. Totally, 363 DE-CRGs were selected, and GRAP2 and TREM2 were identified as key genes for RSA, which were both upregulated in RSA samples. The two genes showed excellent diagnostic performance in both the training and validation cohorts. GSEA revealed that GRAP2 and TREM2 were involved in immune-related pathways. Immune infiltration analysis showed that GRAP2 and TREM2 had a negative correlation with effector memory CD4 T cells. Drug prediction identified that the two genes had 3 common interacting chemicals, including bisphenol A, tetrachlorodibenzodioxin, and air pollutants. In conclusion, this study identified GRAP2 and TREM2 as key genes for RSA and provided insights into the immune mechanisms underlying the condition.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-026-00901-3.

Steroid-induced osteonecrosis of the femoral head (SONFH) is characterized by devastating collapse of the femoral head. Pulsed radiofrequency therapy (PRF) has been widely used in different pathophysiological conditions. Here, we investigated the therapeutic benefit of PRF in SONFH and investigated the role of neurotrophins in the treatment effect of PRF therapy. The beneficial effect of PRF therapy in SONFH patients and the rat model of SONFH was evaluated. The effect of PRF therapy on the levels of neutrotropic factors was examined by ELISA. The functional engagement of nerve growth factor (NGF) in alleviating SONFH progression was investigated in the bone marrow cells and animal model. PRF alleviated SONFH progression in the patients and rat model. PRF therapy increased the plasma levels of NGF in SONFH patients and rat model. NGF treatment prevented Dex-induced cell death and steatosis in bone marrow cells, promoted the expression of osteogenic markers, and ameliorated SONFH progression in the rat model. NGF neutralization counteracted the effect of PRF therapy in the rat model of SONFH. Our findings suggest that PFR therapy ameliorates SONFH progression by promoting NGF production.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00871-y.

Long non-coding RNAs (lncRNAs) have been recognized as key modulators in stomach adenocarcinoma (STAD). This study investigated the functional and clinical relevance of LINC01614 in STAD. 120 patients with STAD were enrolled. The levels of LINC01614 and miR-129-2-3p were quantified by qRT-PCR. Prognostic relevance was evaluated using Kaplan-Meier curve and Cox regression. The regulatory relationship of molecular was assessed via Pearson correlation, bioinformatic prediction, and dual-luciferase reporter assays. Proliferation and motility were evaluated through CCK-8 and Transwell assays, with co-transfection experiments performed to assess their functional interplay. LINC01614 was upregulated, whereas miR-129-2-3p was reduced in both STAD tissues and cell lines. Elevated LINC01614 levels were strongly related to advanced pathological TNM, lymph node metastasis, and reduced survival prognostic, identifying it as an independent prognostic marker. Dual-luciferase assays confirmed that LINC01614 directly binds to miR-129-2-3p, and their expression levels exhibited a significant inverse correlation. Functionally, silencing LINC01614 inhibited cell proliferation, migration, and invasion, while co-transfection with a miR-inhibitor partially reversed these cellular activities. LINC01614 is a potential marker of prognosis in STAD and propels tumorigenesis through its interaction with miR-129-2-3p. LINC01614 may represent a novel molecular target for STAD.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00870-z.

SLC44A4 is involved in the transmembrane transport of choline and other organic cations, influencing phospholipid metabolism in cell membranes and potentially participating in immune regulation. Ovarian clear cell carcinoma (OCCC), often diagnosed at advanced stages, exhibits poor responsiveness to conventional surgery and chemotherapy. Therefore, there is an urgent need to identify new biomarkers that can provide insights into the mechanisms underlying its onset and progression.To investigate the impact of SLC44A4 overexpression on the biological behavior of OCCC cells and to explore the underlying mechanisms.The effects of SLC44A4 on the proliferation, migration, and invasion of OCCC cells were assessed using CCK-8, colony formation, scratch, and Transwell assays. The impact of SLC44A4 on the extracellular matrix and PI3K/Akt signaling pathway was evaluated by Western blotting.SLC44A4 inhibited the proliferation and migration of OCCC cells in vitro, altered the extracellular matrix, and may inhibit the PI3K/Akt signaling pathway.SLC44A4 overexpression can alter the tumor microenvironment and inhibit the malignant progression of ovarian clear cell carcinoma (OCCC) by remodeling its extracellular matrix. These findings suggest that SLC44A4 may serve as a potential biomarker for evaluating the prognosis of OCCC.

Delayed fracture healing impairs patient quality of life, with emerging evidence highlighting long non-coding RNAs (lncRNAs) as pivotal regulators in this process. LncRNA NNT-AS1 was up-regulated in delayed fracture healing, but its mechanism remains unclear. This study analysed 97 controls with normal healing and 106 cases of delayed fracture healing. RT-qPCR was employed to assess the levels of NNT-AS1, microRNA-181a-5p (miR-181a-5p), and osteoblast markers (RUNX-2, ALP, OPG) in serum or MC3T3-E1 cells. Detection of osteoblast markers and downstream target genes (BMP3) at the protein level via Western blot analysis. Evaluation of NNT-AS1's diagnostic potential in identifying delayed fracture healing through ROC curve analysis. The interaction between NNT-AS1 and miR-181a-5p was validated using dual luciferase reporter assays and RNA immunoprecipitation. Cell viability was assessed via CCK-8 proliferation assays. Clinical data indicated that serum NNT-AS1 levels were elevated in patients with delayed fracture healing, with high diagnostic value as determined by ROC curve analysis. In vitro experiments demonstrated that NNT-AS1 expression decreased during osteogenic differentiation, and its overexpression inhibited osteoblast activity and osteogenic differentiation. Mechanistically, NNT-AS1 may act as a competitive messenger RNA directly targeting miR-181a-5p. Serum miR-181a-5p was down-regulated in delayed healing patients and negatively correlated with NNT-AS1. And miR-181a-5p mimic reversed the inhibitory effects of NNT-AS1 overexpression on osteogenic differentiation and cell viability. BMP3 may be a target gene downstream of the NNT-AS1/miR-181a-5p axis. NNT-AS1 sponged miR-181a-5p to inhibit osteogenesis, serving as a delayed fracture healing biomarker and therapeutic target.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00887-4.