Anastasis is a phenomenon described as a cellular escape from ethanol-induced cell death. Although the relevant mechanism has not yet been fully elucidated, anastasis is thought to play a role in drug resistance in cancer cells. To date, the regulation of anastasis in normal and cancerous cells has not been clarified. The current cancer treatment strategies are expected to selectively attack cancer cells without negatively affecting normal cell proliferation. Inspired by the anti-cancer potential of bee venom, this study is the first to evaluate whether bee venom has similar selectivity in producing an anastatic effect. The results indicated that bee venom induces anastasis in normal cells (Michigan Cancer Foundation-10A (MCF10A), Adult Retinal Pigment Epithelium cell line-19 (ARPE-19), and National Institutes of Health 3T3 cell line (NIH3T3)) but causes irreversible cell death in breast cancer cells (M.D. Anderson-Metastatic Breast-231 (MDA-MB-231) and Michigan Cancer Foundation-7 (MCF7)). Liver cancer (HepG2) cells were moderately more resistant to permanent cell death after bee venom treatment compared to breast cancer cells. However, cisplatin caused permanent non-selective cell death in both normal and cancerous cells. The selectivity indices after bee venom treatment were higher compared to cisplatin. Taken together, bee venom was shown to induce selective anastasis only in normal cells, not in cancer cells, which suggests that bee venom has significant potential in selective cancer therapy, especially for breast cancer, via promoting the recovery and maintenance of viability of normal cells.
Bee Venoms/pharmacology* ; Humans ; Animals ; Mice ; Cell Survival/drug effects* ; Breast Neoplasms/pathology* ; Female ; Cell Line, Tumor ; NIH 3T3 Cells ; Antineoplastic Agents/pharmacology* ; Cisplatin/pharmacology* ; Cell Death/drug effects* ; Hep G2 Cells ; MCF-7 Cells

OBJECTIVE: To investigate the expression and physiological significance of the ferroptosis marker 4-hydroxynonenal (4-HNE) in myofibroblasts induced by transforming growth factor-β1 (TGF-β1), providing theoretical evidence for its potential role in the diagnosis and treatment of fibrosis in systemic sclerosis (SSc). METHODS: Mouse embryonic fibroblasts (NIH3t3) were cultured and divided into two groups after 12 h of starvation: the control group (cultured in 1% serum-containing medium) and the TGF-β1 group (cultured in 10 μg/L TGF-β1 with 1% serum-containing medium). Cell morphology changes in both groups were observed under a microscope. To confirm successful establishment of the SSc cell model, fibrosis markers were analyzed using reverse transcription quantitative real-time PCR (RT-qPCR) and Western blot. Next, flow cytometry was employed to assess the intracellular levels of reactive oxygen species (ROS) in both groups. Finally, Western blot and immunofluorescence staining were used to measure the expression of 4-HNE in the TGF-β1-treated cells. RESULTS: Microscopic observations revealed that TGF-β1 treatment caused the NIH3t3 cells to transition from a typical spindle shape to a flat, polygonal shape with multiple protrusions, indicating fibroblast activation. The RT-qPCR and Western blot analyses showed that the expression of the fibrosis marker Vimentin was significantly upregulated in the TGF-β1 group compared with the control group (P < 0.01), confirming that TGF-β1 effectively promoted fibrosis-related gene and protein expression. Flow cytometry results indicated that TGF-β1 significantly elevated intracellular ROS levels, suggesting the induction of oxidative stress. Furthermore, both Western blot and immuno-fluorescence staining demonstrated a significant increase in 4-HNE expression in the TGF-β1-treated cells (immunofluorescence intensity P < 0.05). CONCLUSION TGF-β1 promotes fibroblast activation and fibrosis while inducing ROS production, leading to a marked increase in 4-HNE expression. Given the role of 4-HNE as a marker of lipid peroxidation and its elevated levels in the SSc cell model, this study suggests that 4-HNE could serve as a potential biomarker for fibrosis in SSc. The findings highlight the importance of investigating the mechanisms of 4-HNE in fibrosis and suggest that targeting this pathway could offer new therapeutic opportunities for treating SSc.
Animals ; Mice ; Scleroderma, Systemic/pathology* ; Aldehydes/pharmacology* ; Transforming Growth Factor beta1/metabolism* ; NIH 3T3 Cells ; Ferroptosis ; Reactive Oxygen Species/metabolism* ; Fibrosis ; Fibroblasts/metabolism* ; Biomarkers/metabolism* ; Myofibroblasts/metabolism*

Metallothionein (MT) plays a significant role in heavy metal removal, antioxidant defense, and immune regulation. The current predominant approach for obtaining natural MT is extraction from tissue, which often entails complex procedures resulting in limited yields. In recent years, researchers have adopted the strategy of fusing labels such as GST or His for the heterologous expression of MT. However, a challenge in industrial production arises from the subsequent removal of these labels, which often leads to a significant reduction in the yield. The fusion with elastin-like polypeptides (ELPs) offers a promising solution for achieving soluble expression of the target protein, while providing a simple and fast purification process. In this study, ELP was fused with MT, which significantly up-regulated the soluble expression of MT. The fusion protein ELP-MT with the purity above 97% was obtained efficiently and simply by inverse transition cycling (ITC). ELP-MT exhibited a remarkable 2,2'-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) ammonium salt (ABTS) scavenging activity, with the half maximal inhibitory concentration (IC₅₀) of 0.77 μmol/L, which was 53.7 times that of the vitamin E derivative Trolox. In addition, the fusion protein demonstrated strong 1,1-diphenyl-2-trinitrohydrazine (DPPH) scavenging ability. Furthermore, ELP-MT had no toxicity to the proliferation and promoted the adhesion and migration of NIH/3T3 cells. All these results indicated that ELP-MT had good biocompatibility. We constructed the fusion protein ELP-MT combining the unique properties of MT and elastin, laying a technical foundation for the large-scale production of recombinant MT and facilitating the applications in food, health supplement, and cosmetic industries.
Metallothionein/metabolism* ; Elastin/chemistry* ; Recombinant Fusion Proteins/pharmacology* ; Mice ; Animals ; Peptides/metabolism* ; Escherichia coli/metabolism* ; NIH 3T3 Cells

More than 85% of patients with uveal melanoma (UM) carry a GNAQ or GNA11 mutation at a hotspot codon (Q209) that encodes G protein α subunit q/11 polypeptides (Gα_q/11). GNAQ/11 relies on palmitoylation for membrane association and signal transduction. Despite the palmitoylation of GNAQ/11 was discovered long before, its implication in UM remains unclear. Here, results of palmitoylation-targeted mutagenesis and chemical interference approaches revealed that the loss of GNAQ/11 palmitoylation substantially affected tumor cell proliferation and survival in UM cells. Palmitoylation inhibition through the mutation of palmitoylation sites suppressed GNAQ/11^Q209L-induced malignant transformation in NIH3T3 cells. Importantly, the palmitoylation-deficient oncogenic GNAQ/11 failed to rescue the cell death initiated by the knock down of endogenous GNAQ/11 oncogenes in UM cells, which are much more dependent on Gα_q/11 signaling for cell survival and proliferation than other melanoma cells without GNAQ/11 mutations. Furthermore, the palmitoylation inhibitor, 2-bromopalmitate, also specifically disrupted Gα_q/11 downstream signaling by interfering with the MAPK pathway and BCL2 survival pathway in GNAQ/11-mutant UM cells and showed a notable synergistic effect when applied in combination with the BCL2 inhibitor, ABT-199, in vitro. The findings validate that GNAQ/11 palmitoylation plays a critical role in UM and may serve as a promising therapeutic target for GNAQ/11-driven UM.
Humans ; Mice ; Animals ; Lipoylation ; NIH 3T3 Cells ; Uveal Neoplasms/genetics* ; Melanoma/genetics* ; Cell Proliferation ; Proto-Oncogene Proteins c-bcl-2 ; GTP-Binding Protein alpha Subunits, Gq-G11/genetics*

The aim of this study was to evaluate the biological efficacy of a unique perpendicular protrusion of type-I collagen (Col-I) from TiO
Animals ; Cell Adhesion ; Collagen Type I ; Mice ; NIH 3T3 Cells ; Nanotubes ; Surface Properties ; Titanium

BACKGROUND: Tissue engineering represents a promising approach for the production of bone substitutes. The use of perfusion bioreactors for the culture of bone-forming cells on a three-dimensional porous scaffold resolves mass transport limitations and provides mechanical stimuli. Despite the recent and important development of bioreactors for tissue engineering, the underlying mechanisms leading to the production of bone substitutes remain poorly understood. METHODS: In order to study cell proliferation in a perfusion bioreactor, we propose a simplified experimental set-up using an impermeable scaffold model made of 2 mm diameter glass beads on which mechanosensitive cells, NIH-3T3 fibroblasts are cultured for up to 3 weeks under 10 mL/min culture medium flow. A methodology combining histological procedure, image analysis and analytical calculations allows the description and quantification of cell proliferation and tissue production in relation to the mean wall shear stress within the bioreactor. RESULTS: Results show a massive expansion of the cell phase after 3 weeks in bioreactor compared to static control. A scenario of cell proliferation within the three-dimensional bioreactor porosity over the 3 weeks of culture is proposed pointing out the essential role of the contact points between adjacent beads. Calculations indicate that the mean wall shear stress experienced by the cells changes with culture time, from about 50 mPa at the beginning of the experiment to about 100 mPa after 3 weeks. CONCLUSION: We anticipate that our results will help the development and calibration of predictive models, which rely on estimates and morphological description of cell proliferation under shear stress.
Bioreactors ; Bone Substitutes ; Calibration ; Cell Proliferation ; Fibroblasts ; Glass ; Methods ; NIH 3T3 Cells ; Perfusion ; Porosity ; Tissue Engineering

BACKGROUND: Adipocytes in the tumor microenvironment may provide the metabolic fuel or signal transduction through media and other means to promote a variety of malignant proliferation and invasion, of tumor cells, but their role in lung cancer progression is still unclear. The purpose of this study was to investigate the effect of adipocytes on lung cancer cell biology. METHODS: 3T3-L1 pre-adipocytes were induced into mature adipocytes. The cell morphology was observed by microscopy and Oil Red O staining. MTT assay, colony formation assay, wound-healing and Transwell methods were used to detect lung cancer cell proliferation, migration and invasion ability. The content of triglyceride in cells was determined by colorimetry. RESULTS: The morphology of lung adenocarcinoma A549 cells became more slender after co-culture with mature adipocytes, and the proliferation and cloning ability were significantly enhanced (P<0.05). In addition, mature adipocytes can also promote the migration ability (P<0.05), invasion ability (P<0.01) and accumulation of intracellular lipid (P<0.05) of A549 cells. CONCLUSIONS These findings suggested that adipocytes in tumor microenvironment can promote the proliferation, migration and invasion of lung adenocarcinoma A549 cells, which may be related to lipid metabolism.
A549 Cells ; Adenocarcinoma ; metabolism ; pathology ; physiopathology ; Adenocarcinoma of Lung ; Adipocytes ; cytology ; metabolism ; Animals ; Cell Movement ; Cell Proliferation ; Humans ; Lung Neoplasms ; metabolism ; pathology ; physiopathology ; Mice ; NIH 3T3 Cells ; Triglycerides ; metabolism ; Tumor Microenvironment

Senescence-associated secretory phenotype (SASP) is often a concomitant result of cell senescence, embodied by the enhanced function of secretion. The SASP factors secreted by senescent cells include cytokines, proteases and chemokines, etc, which can exert great influence on local as well as systemic environment and participate in the process of cell senescence, immunoregulation, angiogenesis, cell proliferation and tumor invasion, etc. Relative to the abundance of SASP models in human cells, the in vitro SASP model derived from mouse cells is scarce at present. Therefore, the study aimed to establish a mouse SASP model to facilitate the research in the field. With this objective, we treated the INK4a-deficient mouse NIH-3T3 cells and the wildtype mouse embryonic fibroblasts (MEF) respectively with mitomycin C (MMC), an anticarcinoma drug which could induce DNA damage. The occurring of cell senescence was evaluated by cell morphology, β-gal staining, integration ratio of EdU and Western blot. Quantitative RT-PCR and ELISA were used to detect the expression and secretion of SASP factors, respectively. The results showed that, 8 days after the treatment of NIH-3T3 cells with MMC (1 μg/mL) for 12 h or 24 h, the cells became enlarged and the ratios of β-gal-positive (blue-stained) cells significantly increased, up to 77.4% and 90.4%, respectively. Meanwhile, the expression of P21 protein increased and the integration ratios of EdU significantly decreased (P < 0.01). Quantitative RT-PCR detection showed that the mRNA levels of several SASP genes, including IL-6, TNF-α, IL-1α and IL-1β increased evidently. ELISA detection further observed an enhanced secretion of IL-6 (P < 0.01). On the contrary, although wildtype MEF could also be induced into senescence by MMC treatment for 12 h or 24 h, embodied by the enlarged cell volume, increased ratios of β-gal-positive cells (up to 71.7% and 80.2%, respectively) and enhanced expression of P21 protein, the secretion of IL-6 displayed no significant change. Our study indicated that, although MMC could induce senescence in both mouse NIH-3T3 cells and wildtype MEF, only senescent NIH-3T3 cells displayed the canonical SASP phenomena. Current study suggested that senescent NIH-3T3 cells might be an appropriate in vitro SASP model of mouse cells.
Animals ; Cell Proliferation ; Cellular Senescence ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Cytokines ; genetics ; metabolism ; DNA Damage ; Fibroblasts ; drug effects ; Interleukin-6 ; secretion ; Mice ; Mitomycin ; pharmacology ; NIH 3T3 Cells ; Phenotype

OBJECTIVETo investigate the effects of Zuogui pill, Yougui pill and the relative compositions on the differentiation towards germ cells of stem cells.

METHODThe rat drug sera for Zuogui pill, Yougui pill and the common composition of Zuogui pill and Yougui pill were prepared respectively as the experimental drugs; the mouse embryonic stem cell 1B10 (MESC-1B10) was used as the representative of stem cells; the above rat drug sera were used to intervene MESC-1B10 and the process was traced by microscopy imaging; after 72 h of the intervention, the RNAs were extracted from the different intervened MESC-1B10, cDNAs were synthesized immediately and finally the Real-time quantitative PCR (qPCR) was used to measure the expression patterns of the 10 reproductive-differentiation-related genes for each intervention.

RESULTThe rat drug serum of Zuogui pill (ZGW-RS) significantly up-regulated Oct-4 and SCP3 and significantly down-regulated GDF-9 and Stra8; the rat drug serum of Yougui pill (ZGW-RS) significantly up-regulated Oct-4, GDF-9, Mvh and SCP3 and significantly down-regulated Stra8, Itga6 and Itgb1; the rat drug sera for the common composition of Zuogui pill and Yougui pill (ZGWYGW-RS) significantly up-regulated Oct-4, SCP3 and ZP3 and significantly down-regulated GDF-9, Stra8, Itga6 and Itgb1.

CONCLUSIONZGW-RS can initiate the change towards meiosis, but can not start the reproductive differentiation of MESC-1B10; YGW-RS can initiate the change towards meiosis, and can also start the reproductive differentiation of MESC-1B10 towards female germ cells; ZGWYGW-RS can initiate the change towards meiosis, and can lightly start the reproductive differentiation of MESC-1B10 towards female germ cells but the inductive effect is smaller than YGW-RS. The experimental results, on one hand, strengthen the knowledge about the influence of the relative compositions of Zuogui pill and Yougui pill on the reproductive differentiation of stem cells, on the other hand, help to explain the mechanism of the treatment of the infertility by Zuogui pill and Yougui pill.

Animals ; Cell Differentiation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Embryonic Stem Cells ; cytology ; drug effects ; Female ; Germ Cells ; cytology ; drug effects ; Infertility ; drug therapy ; Male ; Mice ; NIH 3T3 Cells ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of nickel-smelting fumes on the expression of bcl-2 and bax in mammalian cells.

METHODSLogarithmic growth NIH/3T3 cells were exposed to venom for 24 h, which sample fumes concentration was respectively 0, 6.25, 12.50, 25.00, 50.00, 100.00 µg/ml. Cell viability was assessed by MTT assay and the level of extracellular LDH activity was detected with Lactate Dehydrogenase (LDH) kit. Morphological changes of apoptotic were observed with Hoechst33342, while Western blot was used to measure the expression of bcl-2 and bax.

RESULTSIn addition to 7 days of 6.25 µg/ml nickel-smelting fumes group, each time point and dose group's cell viability reduced with significant differences compared with the control group (P < 0.05). the extracellular LDH activity increased with increasing dose of nickel-smelting fumes, and the extracellular LDH activity of 6.25, 12.50, 25.00, 50.00, 100.00 µg/ml nickel-smelting fumes group increased as compared with the control group (P < 0.05). Simultaneously, the cells, treated with 100.00 µg/ml nickel-smelting fumes for 24 h, appeared obvious morphological changes of apoptosis, such as chromatin condensation and cell shrinkage. the expression of bcl-2 significantly increased in groups of 6.25, 12.50, 25.00 µg/ml nickel-smelting fumes (0.58 ± 0.01, 0.6 3± 0.01 and 0.57 ± 0.01) and decreased in groups of 50.00, 100.00 µg/m nickel-smelting fume (0.35 ± 0.01 and 0.27 ± 0.01) as compared with that of the control group (P < 0.05). And the expression of bax significantly decreased in group of 6.25 µg/ml nickel-smelting fumes (0.58 ± 0.00) and increased in groups of 50.00, 100.00 µg/m nickel-smelting fumes (0.71 ± 0.01 and 0.78 ± 0.02) as compared with that of the control group (P < 0.05).

CONCLUSIONApoptosis was activated in NIH/3T3 cell after 24 h of exposure to Ni-smelting fumes, which may be induced by oxidative stress.

Air Pollutants ; toxicity ; Animals ; Apoptosis ; drug effects ; Cell Survival ; drug effects ; L-Lactate Dehydrogenase ; Mice ; NIH 3T3 Cells ; drug effects ; Nickel ; toxicity ; Oxidative Stress ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism