Deoxynivalenol induces the p21-mediated G₂/M arrest and upregulation of differentiation markers in human dental pulp stem cells.

Mechanistic insights into deoxynivalenol-induced cytotoxicity in an SH-SY5Y 3D spheroid model.

Deoxynivalenol (DON) is a trichothecene mycotoxin frequently detected in cereal-derived foods and animal feed. Exposure to DON has been associated with a variety of toxic effects, including neurotoxicity. Three-dimensional (3D) cell culture systems have emerged as more physiologically relevant models than conventional two-dimensional (2D) cultures, as they better reproduce cellular organization, including cell-cell and cell-matrix interactions, and more accurately reflect in vivo conditions. In this context, the present study aimed to investigate the cytotoxic effects of DON and its underlying mechanisms using a 3D spheroid model derived from the human neuroblastoma cell line SH-SY5Y. Spheroids were exposed to different concentrations of DON and cell viability was assessed using the MTT assay, yielding an IC₅₀ value > 64µM. No significant changes were detected in total intracellular reactive oxygen species (ROS) levels. However, a significant increase in mitochondrial superoxide production was observed at 16µM, together with alterations in mitochondrial membrane potential between 2 and 16µM. In addition, DON treatment altered cell cycle progression and increased the proportion of early apoptotic cells at 16µM, as determined by Annexin V/propidium iodide staining. Protein analysis further revealed increase expression of Bax and Bcl-2 at 16µM, resulting in a Bax/Bcl-2 ratio close to unity, along with elevated levels of cleaved caspase-3. Overall, these results demonstrate that DON induces cytotoxic effects in SH-SY5Y spheroids, accompanied by mitochondrial alterations, increased mitochondrial superoxide production, and apoptotic cell death. These findings contribute to the understanding of DON-induced alterations in neuronal cells and highlight the potential of SH-SY5Y spheroids as a biologically relevant human in vitro model for mechanistic studies of mycotoxin toxicity.

Naphthoquinones mediate differentiation of human umbilical cord derived mesenchymal stem cells into insulin producing cells through regulation of Wnt and BMP pathways

Introduction: Diabetes mellitus (DM) being a chronic metabolic disorder, causes a major concern for the healthcare system. Among different types, type 1 DM (T1DM) results in the destruction of insulin-producing pancreatic β cells, mediated by the immune system. Studies have demonstrated that human umbilical cord derived mesenchymal stem cells (hUMSCs) exhibit great potential to regenerate β-cells. Moreover, in order to enhance the regenerative potential of MSCs, several strategies are being utilized, including preconditioning with bioactive compounds. Among these, naphthoquinones can be used for MSC preconditioning in order to augment their therapeutic potential for β-cell regeneration, as these compounds possess anti‐inflammatory and anti-diabetic properties. Methods: hUMSCs were isolated, characterized, and treated with non-cytotoxic concentrations of lawsone, lapachol, or their combination. The preconditioned cells were subsequently analyzed for pancreatic β-cell differentiation at gene and protein levels. The study also explores the role of Wnt and BMP signaling pathways during the differentiation process through gene expression analysis. Binding patterns of these compounds with their respective receptors were analyzed using in silico studies. Results: Gene expression profiling showed overexpression of pancreatic β-cell–specific markers in the Law + hUMSC group, whereas downregulation of Neurogenin-3 (NGN3) was observed in all treatment groups. Immunocytochemical analysis also showed enhanced expression of insulin in Law + hUMSCs, relative to other groups. Transcriptional analysis of the wingless/integrated (Wnt) and bone morphogenetic protein (BMP) pathways showed increased Wnt and decreased BMP expression across all treatment groups. In silico analyses showed that the binding patterns of lawsone or lapachol with frizzled (FZD) and activin-like kinase 1 (ALK1) receptors share comparable sequence similarity, facilitating their binding to these receptors and regulating downstream Wnt and BMP signaling. Conclusion: The study concludes that the regulatory role of lawsone and lapachol can be exploited for preconditioning of MSCs for improved pancreatic β-cell differentiation.

Homoharringtonine in combination with Tanshinone IIA suppresses the progression of lymphoma cells by downregulating the Akt/MMP signaling pathway

[Cytochalasin B-Induced adipose stem cell-derived nanovesicles promote hair follicle regeneration via activation of the β-catenin signaling pathway].

OBJECTIVES To evaluate the regulatory effects of adipose stem cell (ADSC)-derived cytochalasin B-induced nanovesicles (CINVs) on the biological functions of dermal papilla cells (DPCs) and on hair follicle cycle transition, and to explore the underlying molecular mechanisms. METHODS CINVs were prepared from ADSCs. Their effects on DPC proliferation, migration, and apoptosis were assessed in vitro. A mouse depilation model was used to evaluate the ability of CINVs to promote the transition of hair follicles from telogen to anagen. Western blotting, hematoxylin-eosin (HE) staining, and immunofluo-rescence analyses were performed to assess the activation of β-catenin-related signaling pathways and to elucidate the molecular mechanisms of CINV-mediated hair follicle regeneration. RESULTS CCK-8 assays showed that CINVs at 40 and 60 μg/mL significantly enhanced DPC proliferation. Wound healing assays (at 40 μg/mL) confirmed that CINVs markedly promoted DPC migration. Western blotting revealed that, compared with the control group, CINV treatment significantly increased the expression of phosphorylated GSK-3β (p-GSK-3β, P<0.05) and upregulated β-catenin, the core effector of the canonical Wnt/β-catenin pathway (P<0.01). Moreover, cyclin D1 (a positive regulator of cell cycle progression) expression was elevated (P<0.05), and the levels of phosphorylated AKT (p-AKT, associated with cell survival) and phosphorylated ERK (p-ERK, associated with cell proliferation) were significantly increased (both P<0.05). The Bcl-2/Bax ratio (an anti-apoptotic index) was also increased. In vivo, CINV-treated mice displayed darkened dorsal skin and enlarged hair bulb diameters, along with elevated proportions of β-catenin- and Ki67-positive cells. These effects were significantly attenuated by co-administration of the β-catenin inhibitor MSAB. CONCLUSIONS ADSC-derived CINVs effectively regulate the biological functions of DPCs and promote the transition of hair follicles from telogen to anagen, primarily through activation of the β-catenin signaling pathway.

Dihydroaustrasulfone Alcohol Induces Biphasic Autophagy and Apoptosis in Leukemia Cells via PI3K/Akt and JNK Signaling Pathways

Background/Aim: Dihydroaustrasulfone alcohol (DA), a synthetic precursor of austrasulfone derived from the soft coral Cladiella australis, has demonstrated cytotoxic activity against various cancer types. However, its antitumor effects and underlying mechanisms in human leukemia cells remain unknown. This study aimed to investigate the anticancer effects of DA in leukemia cell models and to elucidate its mechanisms of action. Materials and Methods: Human leukemia cell lines U937 and HL-60 were treated with DA (0–50 μM) for 24 and 48 h. Cell viability was assessed utilizing the Cell Counting Kit-8 (CCK-8) assay. Apoptosis and cell cycle distribution were examined through flow cytometry using Annexin V/PI and PI/RNase staining, respectively. The expression levels of proteins related to apoptosis, autophagy, and signaling pathways were determined via western blot analysis. Results: DA decreased cell viability in a dose- and time-dependent manner. The IC50 values for U937 cells were 15.14 μM and 13.6 μM at 24 and 48 h, respectively; for HL-60 cells, these values were 19.36 μM and 15.93 μM. DA induced SubG1-phase cell cycle arrest (11.6% at 24 h; 39.9% at 48 h) and enhanced apoptosis, as evidenced by increased Bax and PARP-1 cleavage and decreased Bcl-2 expression. At 24 h, DA temporarily induced autophagy, as shown by elevated LC3-II/LC3-I ratios and reduced p62 and Atg5 levels; however, autophagic flux was suppressed at 48 h. Furthermore, DA inhibited phosphorylation of PI3K p85 at 24 h and activated JNK phosphorylation at both time points. Conclusion: DA elicits a biphasic cellular response in U937 leukemia cells, characterized by transient cytoprotective autophagy followed by apoptotic cell death mediated through dysregulation of Bcl-2/Bax and suppression of the PI3K/Akt pathway. These findings indicate that DA is a promising candidate for novel antileukemic therapeutic strategies.

Enoxaparin induces apoptosis and autophagy, modulates inflammatory signaling, and reduces oxidative DNA damage in breast and liver cancer cells.

Cancer progression involves intricate interactions between inflammatory signaling, programmed cell death mechanisms, and oxidative stress. Although enoxaparin is widely used for managing cancer-associated thrombosis, its direct cellular effects on tumor biology remain insufficiently characterized. This study aimed to evaluate the impact of enoxaparin on apoptosis, autophagy, inflammatory mediators, and oxidative DNA damage in breast (MDA-MB-231) and liver (HepG2) cancer cell lines. MDA-MB-231, HepG2, and non-cancerous HEK-293 cells were treated with varying concentrations (5, 10, 20, 40, and 80 mg/mL) of enoxaparin for 24 and 48 h. Cell viability was assessed using the MTT assay, while apoptosis was quantified by TUNEL analysis. Immunofluorescence staining was employed to evaluate the expression of NF-κB, IL-6, TNF-α, LC3, and p62. Oxidative DNA damage was determined by measuring extracellular 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels using a competitive ELISA. Statistical analyses were conducted to compare the treated and control groups. Enoxaparin significantly reduced cell viability in MDA-MB-231 and HepG2 cells without inducing cytotoxicity in HEK-293 cells. Apoptosis was markedly increased in both cancer cell lines following treatment. Enoxaparin differentially modulated inflammatory signaling; NF-κB expression was significantly increased in MDA-MB-231 cells, accompanied by suppression of IL-6 and TNF-α, whereas no significant inflammatory changes were observed in HepG2 cells. Enoxaparin treatment was observed to increase LC3 and p62 expression in both MDA-MB-231 and HepG2 cells, triggering autophagy-related pathways. Moreover, enoxaparin significantly reduced extracellular 8-OHdG levels, suggesting a reduction in oxidative DNA damage. Enoxaparin exhibits multifaceted anticancer effects by promoting apoptosis and autophagy, selectively modulating inflammatory pathways, and reducing oxidative DNA damage in breast and liver cancer cells.