Cell Biology International最新文献

Reconstruction of injured bone remains challenging in the clinic owing to the lack of suitable bone grafts. The utilization of PAI-1 transfected-conditioned media (P-CM) has demonstrated its ability to facilitate the differentiation process of mesenchymal stem cells (MSCs), potentially serving as a crucial mediator in tissue regeneration. This research endeavored to explore the therapeutic potential of P-CM concerning the differentiation of human bone marrow mesenchymal stem cells (hBMSCs). To assess new bone formation, a rat calvaria critical defect model was employed, while in vitro experiments involved the use of the alizarin Red-S mineral induction test. In the rat calvaria critical defect model, P-CM treatment resulted in significan new bone formation. In vitro, P-CM treated hBMSCs displayed robust osteogenesis compared to the control group, as demonstrated by the mineral induction test using alizarin Red-S. P-CM with hydroxyapatite/β-tricalcium phosphate/fibrin gel treatment significantly exhibited new bone formation, and the expression of osteogenic associated markers was enhanced in the P-CM-treated group. In conclusion, results demonstrate that P-CM treatment significantly enhanced the osteogenic differantiation efficiency and new bone formation, thus could be used as an ideal therapeutic biomolecule for constructing bone-specific implants, especially for orthopedic and dental applications.

Gestational diabetes mellitus (GDM) is a common disorder in the clinic, which may lead to severe detrimental outcomes both for mothers and infants. However, the underlying mechanisms for GDM are still not clear. In the present study, we performed label-free proteomics using placentas from GDM patients and normal controls. Vitronectin caused our attention among differentially expressed proteins due to its potential role in the pathological progression of GDM. Vitronectin was increased in the placentas of GDM patients, which was confirmed by Western blot analysis. Vitronectin represses insulin signal transduction in trophoblast cells, whereas the knockdown of vitronectin further potentiates insulin-evoked events. Neutralization of CD51/61 abolishes the repressed insulin signal transduction in vitronectin-treated trophoblast cells. Moreover, vitronectin activates JNK in a CD51/61-depedent manner. Inhibition of JNK rescues impaired insulin signal transduction induced by vitronectin. Overall, our data indicate that vitronectin binds CD51/61 in trophoblast cells to activate JNK, and thus induces insulin resistance. In this regard, increased expression of vitronectin is likely a risk factor for the pathological progression of GDM. Moreover, blockade of vitronectin production or its receptors (CD51/61) may have therapeutic potential for dealing with GDM.

Apelin and its receptor (APJ) are expressed in the reproductive organs of some mammalian females. The function of oviduct has also been suggested to be compromised in the hyperandrogenism condition. However, expression of apelin and APJ has not been shown in the oviduct of hyperandrogenized mice. Thus, the present study has investigated the localization and expression of apelin and APJ in the letrozole-induced hyperandrogenized mice oviduct. Histomorphometric analysis showed decreased lumen of oviduct in the hyperandrogenized mice. Our results showed elevated expression of APJ and decreased abundance of apelin in the hyperandrogenized mice oviduct. This finding suggests impaired apelin signaling in the oviduct of hyperandrogenized mice. The expression of androgen receptor was upregulated while estrogen receptors were downregulated in the hyperandrogenized mice. The expression of HSP70 was also downregulated along with increased expression of active caspase 3 and BAX and decreased expression of BCL2 in hyperandrogenized mice. Furthermore, the phosphorylation of phospho-Ser473-Akt and phospho-Thr308-Akt also showed differential levels in the oviduct of hyperandrogenized mice. Whether this differential phosphorylation of Akt was solely due to impaired apelin signaling in the oviduct, remains unclear. Moreover, increased androgen signaling and suppressed estrogen signaling coincides with elevated apoptosis. In conclusion, hyperandrogenized conditions could also impair the gamete transport and fertilization process due to apoptosis in the oviduct. However, further study would be required to unravel the exact role of apelin signaling in the oviduct in relation to apoptosis.

Anoikis is a process of programmed cell death induced by the loss of cell/matrix interactions. In previous work, we have shown that the acquisition of anoikis resistance upregulates syndecan-4 (SDC4) expression in endothelial cells. In addition, SDC4 gene silencing by microRNA interference reverses the transformed phenotype of anoikis-resistant endothelial cells. Due to this role of SDC4 in regulating the behavior of anoikis-resistant endothelial cells, we have evaluated that the functional consequences of SDC4 silencing in the extracellular matrix (ECM) remodeling in anoikis-resistant rabbit aortic endothelial cells submitted to SDC4 gene silencing (miR-Syn4-Adh-1-EC). For this, we evaluated the expression of adhesive proteins, ECM receptors, nonreceptor protein-tyrosine kinases, and ECM-degrading enzymes and their inhibitors. Altered cell behavior was monitored by adhesion, migration, and tube formation assays. We found that SDC4 silencing led to a decrease in migration and angiogenic capacity of anoikis-resistant endothelial cells; this was accompanied by an increase in adhesion to fibronectin. Furthermore, after SDC4 silencing, we observed an increase in the expression of fibronectin, collagen IV, and vitronectin, and a decrease in the expression of integrin α5β1 and αvβ3, besides that, silenced cells show an increase in Src and FAK expression. Quantitative polymerase chain reaction and Western blot analysis demonstrated that SDC4 silencing leads to altered gene and protein expression of MMP2, MMP9, and HSPE. Compared with parental cells, SDC4 silenced cells showed a decrease in nitric oxide production and eNOS expression. In conclusion, these data demonstrate that SDC4 plays an important role in ECM remodeling. In addition, our findings represent an important step toward understanding the mechanism by which SDC4 can reverse the transformed phenotype of anoikis-resistant endothelial cells.

During the morphological changes occurring in osteoblast differentiation, Sonic hedgehog (Shh) plays a crucial role. While some progress has been made in understanding this process, the epigenetic mechanisms governing the expression of Hh signaling members in response to bone morphogenetic protein 7 (BMP7) signaling in osteoblasts remain poorly understood. To delve deeper into this issue, we treated pre-osteoblasts (pObs) with 100 ng/mL of BMP7 for up to 21 days. Initially, we validated the osteogenic phenotype by confirming elevated expression of well-defined gene biomarkers, including Runx2, Osterix, Alkaline Phosphatase (Alp), and bone sialoprotein (Bsp). Simultaneously, Hh signaling-related members Sonic (Shh), Indian (Ihh), and Desert (Dhh) Hedgehog (Hh) exhibited nuanced modulation over the 21 days in vitro period. Subsequently, we evaluated epigenetic markers, and our data revealed a notable change in the CpG methylation profile, considering the methylation/hydroxymethylation ratio. CpG methylation is a reversible process regulated by DNA methyltransferases and demethylases, including Ten-eleven translocation (Tets), which also exhibited changes during the acquisition of the osteogenic phenotype. Specifically, we measured the methylation pattern of Shh-related genes and demonstrated a positive Pearson correlation for GLI Family Zinc Finger 1 (Gli1) and Patched (Ptch1). This data underscores the significance of the epigenetic machinery in modulating the BMP7-induced osteogenic phenotype by influencing the activity of Shh-related genes. In conclusion, this study highlights the positive impact of epigenetic control on the expression of genes related to hedgehog signaling during the morphogenetic changes induced by BMP7 signaling in osteoblasts.

Melanoma is an aggressive malignancy and remains a major cause of skin cancer mortality, highlighting the need for new treatment strategies. Recent findings revealed that L-kynurenine and quinolinic acid induce cytotoxicity and morphological changes in B16 F10 melanoma cells in vitro. This paper highlights the effects of L-kynurenine and quinolinic acid at previously determined half-maximal inhibitory concentrations on cell cycle progression, cell death and extracellular signal-regulated protein kinase inhibition. Melanoma, B16 F10 and murine macrophages, RAW 264.7 cells were used in this study, as both cell lines express all the enzymes associated with the kynurenine pathway. Post exposure to the compounds at half-maximal inhibitory concentrations, transmission electron microscopy was used to assess intracellular morphological changes. Flow cytometry was used to analyse cell cycle progression and quantify apoptosis via the dual staining of Annexin V and propidium iodide and cell survival via extracellular signal-regulated protein kinase. L-kynurenine and quinolinic acid at half-maximal inhibitory concentrations induced intracellular morphological changes representative of cell death. Flow cytometry revealed alterations in cell cycle distribution, increased apoptosis and significantly inhibition of cell survival. L-kynurenine and quinolinic acid are exogenous kynurenine compounds which inhibited cell survival through extracellular signal-regulated protein kinase inhibition, induced cell cycle alterations and induced apoptosis in B16 F10 melanoma cells.

Daurisoline (DS) is an isoquinoline alkaloid that exerts anticancer activities in various cancer cells. However, the underlying mechanisms through which DS affects the survival of breast cancer cells remain poorly understood. Therefore, the present study was undertaken to investigate the potential anticancer effect of DS on breast cancer cells and reveal the mechanism underlying the enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by DS. Cell counting kit-8 (CCK-8) and 5-ethynyl-2-deoxyuridine (EdU) assay were used to evaluate the ability of cell proliferation. Flow cytometry was selected to examine the cell cycle distribution. TUNEL assay was used to detect the cell apoptosis. The protein expression was measured by Western blot analysis. DS was found to reduce the cell viability and suppress the proliferation of MCF-7 and MDA-MB-231 cells by causing G1 phase cell cycle arrest. DS could trigger apoptosis by promoting the cleavage of caspase-8 and PARP. The phosphorylation of ERK, JNK, and p38MAPK was upregulated clearly following DS treatment. Notably, SP600125 (JNK inhibitor) pretreatment significantly abrogated DS-induced PARP cleavage. DS inactivated Akt/mTOR and Wnt/β-catenin signaling pathway and upregulated the expression of ER stress-related proteins. Additionally, DS amplified TRAIL-caused viability reduction and apoptosis in breast cancer cells. Mechanismly, DS upregulated the protein level of DR4 and DR5, and knockdown of DR5 attenuated the cotreatment-induced cleavage of PARP. Inhibition of JNK could block DS-induced upregulation of DR5. This study provides valuable insights into the mechanisms of DS inhibiting cell proliferation, triggering apoptosis, and enhancing TRAIL sensitivity of breast cancer cells.

Metabolic reprogramming in cancer occurs due to interaction of cells with the surrounding tumor microenvironment. In the microenvironment of solid tumors, nutrient deprivation is induced by high consumption of nutrients and insufficient vasculature. Tumor cells alter their metabolic strategies to adapt to the microenvironment. To understand the role of these metabolic changes, in the current study, we have mimicked nutrient deprivation condition in vitro to evaluate the associated signaling pathways in breast cancer cells. In our study, we have shown that nutritional deprivation activated p38 MAPK and activating transcription factor-2 (ATF-2) by increased phosphorylation of Thr180/Tyr182 and Thr71, respectively, in breast cancer cells. Pharmacological inhibition of p38 MAPK showed increased cell viability and reduced expression of ATF-2 and RAD23B under nutrient starvation conditions. Further, silencing of ATF-2 showed increased cell viability and decreased expression of RAD23B under nutrient starvation conditions. This suggests the involvement of p38 MAPK/ATF-2/RAD23B axis as a signaling pathway under nutrition starvation in breast cancer cells. The RAD23B mediated proteasome activity was shown to be much higher under stress conditions indicating a crucial role of RAD23B as a target for breast cancer.

Marine molluscan cell lines, required for virus screening and cultivation, form essential tools for developing health management strategies for these animals in the blue economy. Moreover, they are also crucial to develop cultivated seafood. As there is no valid marine molluscan cell line, primary cell cultures are relied upon for all investigations. A sound protocol for generating primary cell cultures from molluscs is entailed, but existing protocols often involve heavy antibiotic usage and depuration that invariably affect gene expression and cell health. This work presents an easy-to-adopt, time-saving protocol using non-depurated mollusc Crassostrea madrasensis, which requires only initial antibiotic treatment and minimal exposure or no use of antibiotics in the cell culture medium. The important experimental considerations for arriving at this protocol have been elucidated. Accordingly, sodium hypochlorite and neomycin sulfate were chosen for disinfecting tissues. The study is the first to use shrimp cell culture medium (SCCM) as a cell culture medium for molluscan cell culture. Despite being osmoconformers, the oysters exhibited stable intracellular osmotic conditions and pH, which, when provided in vitro, promoted effective cardiomyocyte formation. The cell viability could be enhanced using 10% fetal bovine serum (FBS), but healthy cell culture could also be obtained using SCCM without FBS. The optimized culture conditions allowed for regular beating cardiomyocyte clusters that could be retained for a month. Limited cell proliferation, as shown by the BrdU assay, demands further interventions, such as possibly producing induced pluripotent stem cells. The optimized protocol and culture conditions also align with some requirements for producing cultivated meat from marine molluscs.

The limitations of current imaging methods to detect small or superficial endometriotic lesions prompt the search for new molecular targets. TSPO is an 18 KDa protein located in the outer mitochondrial membrane, which can be traced by positron emission tomography (PET) using specific ligands. TSPO is located mostly in neurons and inflammatory sites outside the brain. We hypothesized that it might also be expressed in the human endometrium and endometrial-like tissue, being a target for molecular imaging of endometriosis. This prospective cross-sectional study included 28 women with endometriosis and 11 endometriosis-free controls. Endometriotic lesions (n = 49) and normal peritoneum (n = 13) from endometriosis patients were obtained during laparoscopy, while samples of eutopic endometrium from patients with endometriosis (n = 28) and from control women (n = 11) were collected in the operating room using a flexible device. TSPO mRNA expression was evaluated by quantitative reverse-transcription real-time PCR while protein expression was evaluated by immunohistochemistry with a monoclonal antibody antihuman TSPO. TSPO mRNA expression was detected in an invariable fashion in all tissue types evaluated; however, TSPO protein was found to be more abundant in the glandular epithelium than in the stroma, both in the endometrium and in the endometriotic lesions. Interestingly, hormone therapies did not alter the expression of TSPO, and its presence was mostly negative in tissues adjacent to endometriotic implants. As a proof of concept, the protein expression pattern of TSPO in endometriotic tissue and along the adjacent areas suggests that TSPO-based molecular imaging might be used for noninvasive endometriosis detection.