OBJECTIVE: This study aims to investigate the influence of glucose regulated protein (GRP) 78 on osteoblast differentiation in periodontal ligament fibroblasts (PDLFs) under cyclic mechanical stretch and determine the underlying mechanism.
METHODS: FlexCell 5000 cell mechanical device was applied to simulate the stress environment of orthodontic teeth. GRP78High and GRP78Low subpopulation were obtained by flow sorting. Gene transfection was performed to knockdown GRP78 and c-Src expression and overexpress c-Src. Western blot analysis was used to detect the protein expression of Runt-related gene 2 (RUNX2), Osterix, osteocalcin (OCN), and osteopontin (OPN). Immunoprecipitation assay was used to determine the interaction of GRP78 with c-Src. The formation of cellular mineralized nodules was determined by alizarin red staining.
RESULTS: GRP78 was heterogeneously expressed in PDLFs, and GRP78High and GRP78Low subpopulations were obtained by flow sorting. The osteogenic differentiation ability and phosphorylation level of c-Src kinase in the GRP78High subpopulation were significantly increased compared with those in GRP78Low subpopulation after cyclic mechanical stretch (P<0.05). GRP78 interacted with c-Src in PDLFs. The overexpression c-Src group showed significantly increased osteogenic differentiation ability than the vector group (P<0.05), and the sic-Src group showed significantly decreased osteogenic differentiation ability (P<0.05) after cyclic mechanical stretch.
CONCLUSIONS: GRP78 upregulates c-Src expression by interacting with c-Src kinase and promotes osteogenic differentiation under cyclic mechanical stretch in PDLFs.
目的: 探讨在周期性牵张力作用下葡萄糖调节蛋白78（GRP78）对牙周膜成纤维细胞成骨分化的影响，并阐述其机制。方法: 应用FlexCell 5000细胞应力装置模拟牙齿正畸受力环境；应用流式细胞术细胞分选技术获得牙周膜成纤维细胞GRP78高表达细胞和GRP78低表达细胞；采用基因转染技术敲减GRP78、c-Src的表达以及过表达c-Src；蛋白质印迹实验检测成骨转录因子Runt相关基因2（RUNX2）、锌指结构转录因子（Osterix）以及成骨标志蛋白骨钙蛋白（OCN）、骨桥蛋白（OPN）的表达；免疫共沉淀实验检测GRP78与c-Src激酶的相互作用；茜素红染色实验检测细胞矿化结节的形成。结果: GRP78在牙周膜成纤维细胞呈异质性表达，流式分选实验获得GRP78高表达和GRP78低表达细胞。周期性牵张力处理后，与GRP78低表达细胞相比，GRP78高表达细胞成骨分化能力及c-Src激酶磷酸化水平均升高，差异具有统计学意义（P<0.05）；GRP78与c-Src激酶存在相互作用；与对照组相比，过表达c-Src组细胞成骨分化能力升高（P<0.05），sic-Src组细胞成骨分化能力降低（P<0.05）。结论: GRP78通过与c-Src激酶相互作用并上调其表达，进而促进周期性牵张力诱导的牙周膜成纤维细胞成骨分化。.

Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.

C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through the phosphorylation of their C-tail tyrosine sites, and it regulates various types of cellular activity in association with SFK function. As a cytoplasmic protein, CSK needs be recruited to the plasma membrane to regulate SFKs\' activity. The regulatory mechanism behind CSK activity and its subcellular localization remains largely unclear. In this work, we developed a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) to visualize the CSK activity in live cells. The biosensor, with an optimized substrate peptide, confirmed the crucial Arg107 site in the CSK SH2 domain and displayed sensitivity and specificity to CSK activity, while showing minor responses to co-transfected Src and Fyn. FRET measurements showed that CSK had a relatively mild level of kinase activity in comparison to Src and Fyn in rat airway smooth muscle cells. The biosensor tagged with different submembrane-targeting signals detected CSK activity at both non-lipid raft and lipid raft microregions, while it showed a higher FRET level at non-lipid ones. Co-transfected receptor-type protein tyrosine phosphatase alpha (PTPα) had an inhibitory effect on the CSK FRET response. The biosensor did not detect obvious changes in CSK activity between metastatic cancer cells and normal ones. In conclusion, a novel FRET biosensor was generated to monitor CSK activity and demonstrated CSK activity existing in both non-lipid and lipid raft membrane microregions, being more present at non-lipid ones.

One of the effective therapeutic strategies to treat rheumatoid arthritis (RA)-related bone resorption is to target excessive activation of osteoclasts. We discovered that 6-O-angeloylplenolin (6-OAP), a pseudoguaianolide from Euphorbia thymifolia Linn widely used for the treatment of RA in traditional Chinese medicine, could inhibit RANKL-induced osteoclastogenesis and bone resorption in both RAW264.7 cells and BMMs from 1 μM and protect a collagen-induced arthritis (CIA) mouse model from bone destruction in vivo. The severity of arthritis and bone erosion observed in paw joints and the femurs of the CIA model were attenuated by 6-OAP administered at both dosages (1 or 5 mg/kg, i.g.). BMD, Tb.N and BV/TV were also improved by 6-OAP treatment. Histological analysis and TRAP staining of femurs further confirmed the protective effects of 6-OAP on bone erosion, which is mainly due to reduced osteoclasts. Molecular docking indicated that c-Src might be a target of 6-OAP and phosphorylation of c-Src was suppressed by 6-OAP treatment. CETSA and SPR assay further confirmed the potential interaction between 6-OAP and c-Src. Three signaling molecules downstream of c-Src that are vital to the differentiation and function of osteoclasts, NF-κB, c-Fos and NFATc1, were also suppressed by 6-OAP in vitro. In summary, the results demonstrated that the function of c-Src was disrupted by 6-OAP, which led to the suppression of downstream signaling vital to osteoclast differentiation and function. In conclusion, 6-OAP has the potential to be further developed for the treatment of RA-related bone erosion.

Nonreceptor tyrosine kinase c-Src plays a crucial role in cell signaling and contributes to tumor progression. However, the development of selective c-Src inhibitors turns out to be challenging. In our previous study, we performed posttranslational modification-inspired drug design (PTMI-DD) to provide a plausible way for designing selective kinase inhibitors. In this study, after identifying a unique pocket comprising a less conserved cysteine and an autophosphorylation site in c-Src as well as a promiscuous covalent inhibitor, chemical optimization was performed to obtain (R)-LW-Srci-8 with nearly 75-fold improved potency (IC50 = 35.83 ± 7.21 nM). Crystallographic studies revealed the critical C-F···C═O interactions that may contribute to tight binding. The kinact and Ki values validated the improved binding affinity and decreased warhead reactivity of (R)-LW-Srci-8 for c-Src. Notably, in vitro tyrosine kinase profiling and cellular activity-based protein profiling (ABPP) cooperatively indicated a specific inhibition of c-Src by (R)-LW-Srci-8. Intriguingly, (R)-LW-Srci-8 preferentially binds to inactive c-Src with unphosphorylated Y419 both in vitro and in cells, subsequently disrupting the autophosphorylation. Collectively, our study demonstrated the feasibility of developing selective kinase inhibitors by cotargeting a nucleophilic residue and a posttranslational modification site and providing a chemical probe for c-Src functional studies.

In this work, readily achievable synthetic pathways were utilized for construction of a library of N/S analogues based on the pyrazolopyrimidine scaffold with terminal alkyl or aryl fragments. Subsequently, we evaluated the anticancer effects of these novel analogs against the proliferation of various cancer cell lines, including breast, colon, and liver lines. The results were striking, most of the tested molecules exhibited strong and selective cytotoxic activity against the MDA-MB-231 cancer cell line; IC50 1.13 µM. Structure-activity relationship (SAR) analysis revealed that N-substituted derivatives generally enhanced the cytotoxic effect, particularly with aliphatic side chains that facilitated favorable target interactions. We also investigated apoptosis, DNA fragmentation, invasion assay, and anti-migration effects, and discussed their underlying molecular mechanisms for the most active compound 7c. We demonstrated that 7c N-propyl analogue could inhibit MDA-MB-231 TNBC cell proliferation by inducing apoptosis through the regulation of vital proteins, namely c-Src, p53, and Bax. In addition, our results also revealed the potential of these compounds against tumor metastasis by downregulating the invasion and migration modes. Moreover, the in vitro inhibitory effect of active analogs against c-Src kinase was studied and proved that might be the main cause of their antiproliferative effect. Overall, these compelling results point towards the therapeutic potential of these derivatives, particularly those with N-substitution as promising candidates for the treatment of TNBC type of breast cancer.

BACKGROUND: Cancer cells exhibit selective metabolic reprogramming to promote proliferation, invasiveness, and metastasis. Sphingolipids such as sphingosine and sphinganine have been reported to modulate cell death processes in cancer cells. However, the potential of extracellular sphinganine and its mimetic compounds as inducers of cancer cell death has not been thoroughly investigated.
METHODS: We obtained extracellular conditioned medium from HCT-116 cells treated with the previously reported anticancer composition, goat urine DMSO fraction (GUDF). The extracellular metabolites were purified using a novel and in-house developed vertical tube gel electrophoresis (VTGE) technique and identified through LC-HRMS. Extracellular metabolites such as sphinganine, sphingosine, C16 sphinganine, and phytosphingosine were screened for their inhibitory role against intracellular kinases using molecular docking. Molecular dynamics (MD) simulations were performed to study the inhibitory potential of a novel designed modified mimetic sphinganine (MMS) (Pubchem CID: 162625115) upon c-Src kinase. Furthermore, inhibitory potential and ADME profile of MMS was compared with luteolin, a known c-Src kinase inhibitor.
RESULTS: Data showed accumulation of sphinganine and other sphingolipids such as C16 sphinganine, phytosphingosine, and ceramide (d18:1/14:0) in the extracellular compartment of GUDF-treated HCT-116 cells. Molecular docking projected c-Src kinase as an inhibitory target of sphinganine. MD simulations projected MMS with strong (-7.1 kcal/mol) and specific (MET341, ASP404) binding to the inhibitory pocket of c-Src kinase. The projected MMS showed comparable inhibitory role and acceptable ADME profile over known inhibitors.
CONCLUSIONS: In summary, our findings highlight the significance of extracellular sphinganine and other sphingolipids, including C16 sphinganine, phytosphingosine, and ceramide (d18:1/14:0), in the context of drug-induced cell death in HCT-116 cancer cells. Furthermore, we demonstrated the importance of extracellular sphinganine and its modified mimetic sphinganine (MMS) as a potential inhibitor of c-Src kinase. These findings suggest that MMS holds promise for future applications in targeted and combinatorial anticancer therapy.

Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway.

BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive brain tumor that is common among adults. This aggression is due to increased invasion, migration, proliferation, angiogenesis, and decreased apoptosis. Plant-based compounds have a high potential to be used as an anticancer agent due to their various mechanisms and less undesirable side effects. Potentilla fulgens is a medicinal plant, and methanolic root extract of P. fulgens (PRE) has anti-inflammatory and anticancer properties.
OBJECTIVE: In this study, we aimed to investigate antiproliferative effect of PRE on U118 and T98G glioblastoma cancer cells and to reveal which molecular signaling pathways regulate this mechanism of action.
METHODS: The effect of PRE on cell viability of GBM cells was investigated by MTT assay. Involvement of PRE with cell growth and survival signaling pathways, phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR and c-Src/signal transducer and activator of transcription 3 (STAT3), was examined using Western Blot.
RESULTS: PRE reduced cell viability of GBM and human dermal fibroblast (HDF) cells in a dose-and time-independent manner. PI3K expression/phosphorylation level remained unchanged in both GBM and HDF cells after PRE treatment, but Akt/mTOR signaling pathway was downregulated in PRE-treated cells. PRE treatment did not affect c-Src expression/phosphorylation level in GBM cells; however, expression of c-Src was suppressed in HDF cells. Similar results were observed for STAT3 expression and phosphorylation status.
CONCLUSIONS: PRE has the ability to suppress cell viability in GBM cells, by targeting the Akt/mTOR signaling pathway.

OBJECTIVE: C-terminal Src kinase (CSK), a sarcoma (Src) homologous family kinase, is one of the most important negative regulators. It acts as a tumor suppressor by inhibiting the activity of Src family tyrosine kinases. Paradoxically, CSK is highly expressed in a variety of common tumors. Therefore, we report the expression profile of CSK in pan-cancer patients, focusing on the prognostic value, immune infiltration pattern, and biological function of CSK in gastric cancer.
METHODS: We used the TCGA database to analyze CSK expression, clinical relevance, prognostic significance, assessment of the tumor immune microenvironment, and GO and Kegg enrichment analysis based on co-expressed genes using a bioinformatics approach.
RESULTS: CSK is a protective factor in gastric cancer, and its expression correlates with the level of immune cell infiltration and immune checkpoint molecules.
CONCLUSIONS: Our findings suggest that CSK is an independent prognostic factor in gastric cancer and may predict molecular targeting and immunotherapy and provide ideas for its therapeutic strategy.