Cytotechnology最新文献

Despite improvements in therapeutic approaches, the mortality rate of gastric cancer (GC) remains unacceptably high. Evidence suggests that FXYD domain containing ion transport regulator 6 (FXYD6) is downregulated in GC. However, its exact function and the molecular mechanism in GC are still unclear. FXYD6 expression in different cell lines was estimated using RT-qPCR. Western blotting was employed for protein expression detection. Cell counting kit-8 assay, colony formation assay, and flow cytometry were implemented to assess GC cell viability, proliferation, and apoptosis, respectively. Bioinformatics analysis as well as chromatin immunoprecipitation and luciferase reporter assays were utilized for verifying FXYD6 interaction with the transcription factor Krüppel-like factor 10 (KLF10). The results showed that FXYD6 displayed a decreased level in GC cell lines. Impaired proliferative ability and enhanced apoptotic capacity were observed in GC cells overexpressing FXYD6. KLF10 expression is positively correlated with FXYD6 expression in GC samples. KLF10 binds to the FXYD6 promoter to enhance its transcription. FXYD6 depletion counteracted KLF10 upregulation-triggered reduction in GC cell proliferation and elevation in apoptosis. In conclusion, KLF10 activates FXYD6 transcription, thereby impeding GC cell proliferation and promoting cell apoptosis.

When juxtaposed with 2D cell culture models, multicellular tumor spheroids demonstrate a capacity to faithfully replicate certain features inherent to solid tumors. These include spatial architecture, physiological responses, the release of soluble mediators, patterns of gene expression, and mechanisms of drug resistance. The morphological and behavioural similarities between 3D-cultured cells and cells within tumor masses highlight the potential of these models in studying cancer biology and drug responses. The liquid overlay method, hanging drop technique, and ultra-low adhesion plates are among the various methods for generating tumor spheroids, each with its advantages and applications. Gene expression studies, employing advanced methods such as microarrays, suppression subtractive hybridization, qRT-PCR, and mass-spectrometry-based proteomics revealed distinct expression patterns in 3D spheroids compared to 2D cultures, uncovering upregulation and downregulation of genes associated with tumor development, metastasis, and drug resistance. Protein expression studies identified alterations in key signaling pathways, metabolic characteristics, and phosphorylation levels, highlighting the impact of 3D culture on cellular responses. This study explores genes and proteins expression variations in various cancer cell lines cultivated in 3D spheroids, shedding light on the complexity of interactions in a more tumor-mimicking environment. The fusion of these analytical approaches not only advances scientific understanding but also holds promise for the development of more effective cancer treatments.

Our study probed into how curcumin modulates NF-κB pathway to regulate articular chondrocytes. ATDC5 cells were exposed to varying concentrations of curcumin (0, 10, 20, 50, or 100 μM) for 48 h, followed by an assessment of curcumin's cytotoxicity. Cells were also treated with 10 ng/ml IL-1β, curcumin, 5 μg/L NF-κB inhibitor (PDTC), and 5 μM NLRP3 inflammasome inducer (nigericin) for 48 h, before cell viability, apoptosis, NF-κB pathway-related proteins, NLRP3 inflammasome-related proteins and inflammatory cytokines were detected. IL-1β treatment notably diminished chondrocyte viability and increased apoptosis, evidenced by elevated level of Bax and cleaved caspase-3, and reduced level of Bcl2, while such expression patterns were reversed by curcumin treatment in a concentration-dependent fashion. Additionally, NF-κB pathway and NLRP3 inflammasome in chondrocytes were activated by IL-1β treatment, but can also be suppressed following curcumin intervention. Furthermore, inhibition of NF-κB pathway curtailed the NLRP3 inflammasome activation and chondrocyte apoptosis, while activation of the NLRP3 inflammasome partially reversed the protective impacts of curcumin against chondrocyte apoptosis. Curcumin inhibits NF-κB pathway, thereby preventing the NLRP3 inflammasome activation and ameliorating IL-1β-induced apoptosis in articular chondrocytes.

β-Conglycinin was conjugated with carboxymethyl cellulose (CMC) by using water-soluble carbodiimide to improve its function. Two kinds of CMC differing in average molecular weight (about 1 kDa and 90 kDa) were used to investigate the relationship between molecular weight of conjugated saccharide and saccharide content in the conjugates and degree of functional changes in β-conglycinin. The β-conglycinin-CMC conjugates were purified by dialysis using a dialysis membrane whose molecular weight cutoff is 100 kDa. Composition of the β-conglycinin-low molecular weight (LMW) CMC and β-conglycinin-high molecular weight (HMW) CMC was β-conglycinin: CMC = 1:3.3 and 1:2.1 (weight ratio) respectively which was confirmed by BCA method and phenol sulfuric acid method. Conjugation was confirmed by SDS-PAGE with CBB. Solubility of β-conglycinin in the range of pH4.0-7.0 was much improved by conjugation with both LMW and HMW CMC. Emulsifying property of β-conglycinin at pH5.0 and pH7.0 was much improved by conjugation with HMW CMC and greater improvement was achieved by conjugation with LMW CMC. Immunogenicity of β-conglycinin was decreased by conjugation with LMW CMC.

Angiogenesis is an intricate pathway that involves the formation of new blood capillaries from old, functioning ones. Improper angiogenesis is a feature of numerous maladies, including malignancy and autoimmune disorders. Indole-related derivatives are believed to interfere with the mitotic spindle, inhibiting the multiplication, and invasion of cancerous human cells. 5-bromo-2-(5-(4-nitrophenyl)-4H-1,2,4-triazol-3-yl)-1H-indole (2-NTI) is one of such compounds with outstanding anti-angiogenic, and anti-proliferative properties. To evaluate 2-NTI's antiangiogenic and anti-oxidant activities and potential mechanisms of action in comparison with the standard agent, suramin. The rat aortic ring (RAR) and Chick chorioallantois membrane (CAM) assays were employed to determine antiangiogenic efficacy and dose response, while the DPPH assay estimated free radical scavenging activity. Besides, an MTT test was performed to evaluate antiproliferative activity in HUVECs; however, RT-PCR assessed the gene expression level of VEGF in HCT116 cells. 2-NTI displayed a significant and dose-dependent suppression of angiogenesis (83.04%) at 100 μg/mL concentration versus the negative controls in the RAR assay. 2-NTI also showed no toxicity in the HUVEC cell line, with an IC50 of 876.6 μg/mL, but it significantly reduced the formation of free radicals (IC50 of 135.2 µg/mL) and VEGF gene expression (at doses of 200 and 400 µg/mL) versus the negative controls and suramin. In CAM model, 2-NTI generated considerable blood vessel regression as compared to the negative control. 2-NTI possesses potent anti-angiogenic actions, which might be explained by its profound anti-proliferative and free radical detoxifying activities.

Cervical cancer is one of the most common tumors in women and is a major problem in gynecological health. Studies have shown that Yinjia pills (YJP), a traditional Chinese medicine, can effectively slow the progression of cervical cancer. Therefore, this study mainly explored the molecular mechanism by which YJP delays the progression of cervical cancer. The expression level of PKM2 in cervical cancer was evaluated by the gene expression profiling interactive analysis (GEPIA) database, and the prognostic value of the PKM2 gene was evaluated by the Kaplan‒Meier plotter database. HeLa cervical cancer cells were treated with different concentrations of YJP (2.5, 5, 10, and 20 mg/mL). The levels of the inflammatory factors were detected by ELISA. Cell proliferation activity, migration and invasion were detected by CCK-8 assay, Transwell assays and cell scratch experiment. Apoptosis was detected by flow cytometry. Western blotting was used to detect the expression of proteins. In this study, PKM2 was upregulated in both cervical squamous cell carcinoma (CESC) and endometrial adenocarcinoma tissues, and a Kaplan‒Meier analysis showed that higher PKM2 expression was associated with lower patient survival. YJP inhibited the proliferation, migration and invasion of HeLa cells in a dose-dependent manner, promoted the apoptosis of HeLa cells, and inhibited the expression of inflammatory factors. In addition, YJP inhibited the activation of the JAK/STAT3 pathway and the occurrence of EMT. Knockdown of PKM2 also inhibited the malignant biological behavior of HeLa cells, but overexpression of PKM2 weakened the inhibitory effect of YJP on the malignant biological behavior of HeLa cells. Angoline, a JAK/STAT3 pathway inhibitor, attenuated the effect of PKM2 overexpression on the efficacy of YJP. In conclusion, YJP can inhibit the activation of the JAK/STAT3 pathway by regulating PKM2, thereby inhibiting the malignant biological behavior of HeLa cells.

High mobility group protein N2 (HMGN2) related pathways are involved in chromatin regulation/acetylation. It has been reported to be involved in several types of cancers. A recent sequencing study suggested that HMGN2 might be involved in the progression of hepatocellular carcinoma (HCC). This study aimed to explore the role of HMGN2 in HCC, which has been proven to be involved in the development of HCC. In this study, we collected clinical samples and cultured normal hepatocytes and hepatocellular carcinoma cell lines to detect HMGN2 expression levels using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and western blot assay. Subsequently, to determine the role of HMGN2 in HCC, HMGN2 was overexpressed in HCC cell lines. MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide) assay was used to detect the cell proliferative capacity, and proliferation-related proteins were detected by RT-qPCR and western blot assay. To observe the effect of HMGN2 on cell migration and invasion capacity, Transwell assay was performed. Then, cell apoptosis was detected by flow cytometry, and caspase3 and cleaved-caspase3 were detected using western blot assay. Finally, EMT (epithelial to mesenchymal transition)-related proteins, and matrix metalloproteinase-2 (MMP-2) and MMP-9 expression were detected by RT-qPCR and western blot assay. HMGN2 expression was decreased in HCC tissues as well as in HCC cell lines. After overexpression of HMGN2, MTT results suggested that cell proliferation was decreased, and flow cytometry results showed that the apoptosis level was increased and ki-67 and proliferating cell nuclear antigen (PCNA) expression levels were decreased. On the contrary, cleaved-caspase 3 expression level was increased. HCC cells overexpressing HMGN2 showed a drastic reduction in the number of migrating and invading cells, and the expression levels of MMP-2 and MMP-9 were significantly decreased. Finally, E-cadherin expression was elevated in HCC cells transfected with the HMGN2-plasmid, while N-cadherin showed the opposite result. HMGN2 expression was significantly decreased in patients with HCC. HMGN2 inhibits the malignant behavior of HCC cells and is a potential therapeutic target for HCC.

Bone tissue engineering is a promising approach to overcome the limitations of traditional autograft bone transplantation. Graphene quantum dots (GQDs) have been suggested as an enhancement for osteogenic differentiation. This study aimed to investigate the ability of the fibrin hydrogel scaffold in the presence of graphene quantum dots to promote osteogenic differentiation of human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs). The hWJ-MSCs were isolated from the Wharton's jelly of the human umbilical cord using a mechanical method. Fibrin hydrogel scaffolds were prepared by mixing 15 µl of thrombin solution with fibrinogen solution. GQDs were incorporated into the scaffolds at concentrations of 0, 5, and 10 µg/ml. Cell viability was determined through DAPI staining and the MTT assay. Osteogenic differentiation was assessed by measuring alkaline phosphatase (ALP) activity, quantifying calcium deposition using Alizarin Red S staining, and analyzing the gene expression of BGLAP, COL1A1, Runx-2 and ALP via qPCR. Scanning electron microscopy (SEM) was employed to analyze the scaffold architecture. SEM analysis revealed that the fibrin hydrogel exhibited a suitable architecture for tissue engineering, and DAPI staining confirmed cell viability. The MTT results indicated that the GQDs and fibrin hydrogel scaffold exhibited no cytotoxic effects. Furthermore, the incorporation of GQDs at a concentration of 10 µg/ml significantly enhanced ALP activity, calcium deposition, and the expression of osteogenesis-related genes compared to the control. The findings suggest that the combination of fibrin hydrogel and GQDs can effectively promote the osteogenic differentiation of hWJ-MSCs, contributing to the advancement of bone tissue engineering.

Osteoarthritis (OA) is a common form of arthritis characterized by subchondral bone proliferation and articular cartilage degeneration. Recently, the Nod-like receptor pyrin domain 3 (NLRP3) inflammasome has gained attention due to its association with synovial inflammation in OA. Triptolide (TP), known for its immunosuppressive and anti-inflammatory effects, has been studied in various diseases. However, the specific impact of TP on OA and its underlying mechanism remains largely unexplored. In this study, chondrocytes were treated with a specific concentration of TP, and subsequent analysis through Western blotting and immunofluorescence staining revealed decreased expression levels of MMP-13, NLRP3, Caspase-1, ASC, β-catenin, p-p65, and IκB compared to the model group. ELISA results demonstrated significantly lower levels of IL-1β, IL-18, and TNF-α in the TP treatment group compared to the model group. In addition, triptolide ameliorates the degradation of the extracellular matrix (ECM) by enhancing the expression of collagen-II. In conclusion, our findings suggest that TP exhibits anti-inflammatory effects on chondrocytes in the presence of LPS-induced inflammation by inhibiting the activation of the NLRP3 inflammasome via the Wnt/β-catenin and NF-κB pathway. These results contribute to a better understanding of TP's potential therapeutic benefits in managing OA.

Tumor necrosis factor alpha (TNF-α) is a well-known pro-inflammatory cytokine originally recognized for its ability to induce apoptosis and cell death. However, recent research has revealed that TNF-α also plays a crucial role as a mediator of cell survival, influencing a wide range of cellular functions. The signaling of TNF-α is mediated through two distinct receptors, TNFR1 and TNFR2, which trigger various intracellular pathways, including NF-κB, JNK, and caspase signaling cascades. Both TNFR1 and TNFR2 are expressed in astrocytes, which are specialized glial cells essential for maintaining the structural and functional integrity of the central nervous system (CNS). Astrocytes support neuronal function by regulating brain homeostasis, maintaining synaptic function, and supplying metabolic substrates. In addition, astrocytes are known to secrete a variety of growth factors and neurotrophins, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4/5. These neurotrophins play a critical role in supporting neuronal survival, synaptic plasticity, and myelination within the brain. The present study focuses on the role of TNF-α in modulating neurotrophin expression and secretion in rat cortical astrocytes. We demonstrate that TNF-α induces the upregulation of neurotrophins, particularly NGF and BDNF, in cultured astrocytes. This effect is accompanied by an increase in the expression of their respective receptors (TrkA & TrkB), further suggesting a functional modulation of neurotrophic signaling pathways. Notably, we show that the modulation of neurotrophin expression by TNF-α is mediated via the NF-κB signaling pathway. Additionally, we observed that TNF-α also regulates the secretion levels of NGF and BDNF into the culture media of astrocytes in a dose-dependent manner, indicating that TNF-α can modulate both the production and release of these growth factors. Taken together, our findings highlight a previously underexplored neuroprotective role of TNF-α in astrocytes. Specifically, we propose that TNF-α, through the upregulation of neurotrophins, may contribute to maintaining neuronal health and supporting neuroprotection under disease conditions.