Src Inhibitor Research Articles

Rhamnogalacturonan promotes intestinal mucosal repair through increased cell migration.

Mucosal healing is the primary goal for Inflammatory Bowel Diseases (IBD) treatment. We previously showed the direct beneficial effects of rhamnogalacturonan (RGal) on intestinal epithelial barrier function. Here, we aimed to evaluate the effect of RGal in intestinal epithelial wound healing. Confluent cancer cell lines and colonoid monolayers were wounded, treated with RGal for 48 h and assessed using a live cell imaging system. Proliferation and apoptosis of cells were evaluated using EdU and TUNEL assays, respectively. Antagonists and inhibitors were used to determine the receptor and signaling pathways involved. Female and male mice with DSS-induced colitis were treated orally with RGal for 7 days during the recovery phase. RGal enhanced wound healing in Caco-2, T84 and primary cells by increasing cell migration. Inhibition of pre-transcriptional signaling pathways FAK, Src, PI3K, Rho family, and JNK reversed the RGal-induced wound healing. RNAseq data from Caco-2 and primary cells treated with RGal showed the upregulation of NF-κB pathway at 12 h. Actinomycin D, Bay 11-7082 or JSH-23, and NS-398 treatment significantly reversed the effect of RGal on wound healing, confirming that the response was also transcriptionally dependent and involved NF-κB signaling and downstream COX-2 protein activity. RGal treatment of male mice enhanced recovery from DSS colitis. RGal promoted wound healing in cancer and primary cells by increasing cell migration and accelerated epithelial mucosal healing in male mice. Our findings show a novel mechanism of action of RGal in wound healing that could help in mucosal healing and the resolution of intestinal inflammation.

The impact of signaling pathways on the desmosome ultrastructure in pemphigus

IntroductionThe autoantibody-driven disease pemphigus vulgaris (PV) impairs desmosome adhesion in the epidermis. In desmosomes, the pemphigus autoantigens desmoglein 1 (Dsg1) and Dsg3 link adjacent cells. Dsgs are clustered by plaque proteins and linked to the keratin cytoskeleton by desmoplakin (Dp). The aim of this study was to identify the impact of several PV-related signaling pathways on desmosome ultrastructure.MethodsSTED microscopy, Dispase-based dissociation assay.ResultsAs observed using STED microscopy, pemphigus autoantibodies (PV-IgG) reduced desmosome number, decreased desmosome size, increased plaque distance and thickness and caused loss of adhesion. Decreased desmosome number, increased plaque distance and thickness and loss of adhesion correlate with features found for newly assembled immature desmosomes, observed after Ca2+ depletion and repletion. This was paralleled by plaque asymmetry, keratin filament retraction and fragmentation of Dsg1 and Dsg3 immunostaining. Inhibition of each individual signaling pathway investigated here prevented the loss of adhesion and ameliorated keratin retraction. In addition, inhibition of p38MAPK or PLC completely rescued all parameters of desmosomes ultrastructure and increased desmosome number under basal conditions. In contrast, inhibition of MEK1/2 was only partially protective for desmosome size and plaque thickness, whereas inhibition of Src or increase of cAMP decreased desmosome size but increased the desmosome number even in the presence of PV-IgG. DiscussionAlterations of the desmosomal plaque ultrastructure are closely related to loss of adhesion and regulated differently by signaling pathways involved in pemphigus pathogenesis. This insight may allow identification of novel treatment options targeting specific steps of desmosome turn-over in the future.

IGF2BP3 Triggers STAT3 Pathway by Stabilizing SRC RNA in an m6A-Dependent Manner to Promote Lymphatic Metastasis in LUAD.

Lymph node metastasis significantly affects the NSCLC patients' staging, treatment strategy, and prognosis. Studies have shown that IGF2BP3, an oncofetal protein and an m6A reader, overexpresses and correlates to lymph node metastasis and worse overall survival in histopathological studies including NSCLC, but its mechanism needs further study. This study explored IGF2BP3's function and mechanism in LUAD lymphatic metastasis using public databases, a human LUAD tissue microarray, human LUAD cells, and a lymphatic metastasis model in male BALB/c nude mice. Firstly, we proved that IGF2BP3 overexpression was positively correlated to patients' lymph node metastasis and worse overall survival in bioinformatics and a human LUAD tissue microarray analysis. IGF2BP3 was knocked out or overexpressed in human LUAD cell lines. Functionally, IGF2BP3 facilitated NCI-H1299, NCI-H358, and A549 cell growth, migration, invasion, and EMT invitro, and promoted tumorigenesis, lymphangiogenesis, and lymphatic metastasis of NCI-H1299 cells in BALB/c nude mice. Mechanically, RIP, RNA pull-down assay, MeRIP, mRNA stability assays, rescue experiments, and immunohistochemical assays were conducted. IGF2BP3 was demonstrated to bind to the m6A site of the 3'UTR region of SRC, stabilizing its mRNA and activating the downstream STAT3 signaling pathway and lymphatic growth factors such as VEGF-C, therefore affecting lymphatic metastasis. The cell migration and EMT function of IGF2BP3 were partially rescued by utilizing SRC siRNA or AZD0530, an SRC inhibitor. This study demonstrated that IGF2BP3 promotes lymphatic metastasis in LUAD via activating the m6A-SRC-STAT3-VEGFC signaling axis, indicating that IGF2BP3 is a potential therapeutic target to overcome metastasis in LUAD patients.

Integrating machine learning and structure-based approaches for repurposing potent tyrosine protein kinase Src inhibitors to treat inflammatory disorders

Tyrosine-protein kinase Src plays a key role in cell proliferation and growth under favorable conditions, but its overexpression and genetic mutations can lead to the progression of various inflammatory diseases. Due to the specificity and selectivity problems of previously discovered inhibitors like dasatinib and bosutinib, we employed an integrated machine learning and structure-based drug repurposing strategy to find novel, targeted, and non-toxic Src kinase inhibitors. Different machine learning models including random forest (RF), k-nearest neighbors (K-NN), decision tree, and support vector machine (SVM), were trained using already available bioactivity data of Src kinase targeting compounds. The performance evaluation of these models demonstrated SVM as the best model, which was further utilized to shortlist 51 highly potent compounds by screening an FDA-approved library of 1040 drugs. Molecular docking and molecular dynamic simulation were subsequently employed to evaluate the binding affinity and stability of the proposed compounds. Orlistat, acarbose and afatinib were identified as the potent leads, demonstrating stable conformations and stronger interactions, validated by root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (RoG), and hydrogen bond analyses. Molecular Mechanics/Generalized Born Surface Area (MMGBSA) analysis validated their binding affinities by providing comparably lower binding free energies for orlistat (− 33.4743 ± 3.8908), acarbose (− 19.5455 ± 5.4702), and afatinib (− 36.4944 ± 5.4929) than the control, dasatinib (− 13.7785 ± 5.8058). Finally, toxicity analysis revealed orlistat and acarbose as the possible safer therapeutics by eliminating afatinib as it showed significant toxicity concerns. Our investigation supports the advance computational methods utilization in the field of drug discovery and suggest further experimental validation of proposed inhibitors of Src kinase for their safer use against inflammatory diseases. The ultimate aim of this study is to advance the development of effective treatments for inflammatory diseases, linked with Src overexpression.

SRC enhanced cisplatin resistance in bladder cancer by reprogramming glycolysis and pentose phosphate pathway

The development of cisplatin resistance often results in a grim prognosis in advanced or recurrent bladder cancer. However, effective treatment strategies for cisplatin resistance have not been well established. Herein, we found that overactivation of SRC is associated with cisplatin-resistance. SRC activates hexokinase2 which up-regulates glycolysis and especially the pentose phosphate pathway that leading to increased nucleotide synthesis and NADPH production which can neutralize reactive oxygen species (ROS) induced by cisplatin, thereby protecting bladder cancer cells from cisplatin-induced DNA damage. This phenomenon was effectively reversed by knockout of SRC and inhibition of SRC activity by the SRC inhibitor, eCF506. Moreover, we constructed Cell-derived xenograft (CDX) and Patient-derived xenograft (PDX) from cisplatin-resistant bladder cancer patient. eCF506 exhibited excellent anti-tumor effects and effectively enhanced cisplatin-sensitivity. Altogether, our findings demonstrate that targeting SRC is a promising approach to overcome cisplatin-resistance in bladder cancer, and providing new insights for combination therapy in bladder cancer.

Editorial Expression of Concern: The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

Editorial Expression of Concern: The Src inhibitor dasatinib accelerates the differentiation of human bone marrow-derived mesenchymal stromal cells into osteoblasts

Identification of a novel Src inhibitor K882 derived from quinazoline-based stilbenes with anti-NSCLC effect

Identification of a novel Src inhibitor K882 derived from quinazoline-based stilbenes with anti-NSCLC effect

SRC kinase drives multidrug resistance induced by KRAS-G12C inhibition.

Direct targeting of the KRAS-G12C-mutant protein using covalent inhibitors (G12Ci) acts on human non-small cell lung cancer (NSCLC). However, drug resistance is an emerging concern in this approach. Here, we show that MRTX849, a covalent inhibitor targeting the KRAS-G12C mutation, leads to the reactivation of the mitogen-activated protein kinase signaling pathway in MRTX849-resistant NSCLC and pancreatic ductal adenocarcinoma. A genome-wide CRISPR screen revealed that the adenosine triphosphate binding cassette transporter ABCC1 mediates MRTX849 resistance. Functional studies demonstrated that the transcription factor JUN drives ABCC1 expression, resulting in multidrug resistance. An unbiased drug screen identified the tyrosine kinase inhibitor dasatinib that potentiates MRTX849 efficacy by inhibiting SRC-dependent JUN activation, avoiding multidrug resistance and tumor suppression in vitro as well as in suitable preclinical mouse models and patient-derived organoids. SRC inhibitors (DGY-06-116, dasatinib, and bosutinib) also exhibit synergistic effects with MRTX849 in eliminating various tumor cell lines carrying KRAS-G12C mutations. Thus, SRC inhibitors amplify the therapeutic utility of G12Ci.

N-methyl-D-aspartate receptor activation is downstream coupled to pannexin 1 opening by Src kinase in dorsal horn neurons: an essential link for mechanical hyperalgesia in nerve-injured rats.

A well-recognized molecular entity involved in pain-related neuroplasticity is the N-methyl-D-aspartate receptor (NMDAR), which is crucial for developing chronic pain. Likewise, the pannexin 1 (Panx1) channel has been described as necessary for initiating and maintaining neuropathic pain, driving nociceptive signals dependent on spinal NMDAR through several possible mechanisms. Through behavioral, pharmacological, and molecular approaches, our study in male rats has revealed several key findings: (1) neurons located in spinal cord laminae I and II express functional Panx1 channels in both neuropathic and sham rats. These channels can open (indicated by YOPRO-1 uptake) through the stimulation of NMDARs with intrathecal NMDA; (2) intrathecal NMDA leads to increased expression of pSrc and pPanx1 in dorsal horn neurons. This elevation exacerbates existing mechanical hyperalgesia in nerve-injured rats; (3) inhibition of Src with intrathecal PP2 or blockade of Panx1 with intrathecal 10Panx effectively mitigates NMDA-induced effects and reduces the spontaneous mechanical hyperalgesia of nerve-injured rats. Notably, while 10Panx successfully alleviates hyperalgesia, it does not alter pSrc expression; and (4) NMDA-stimulated YOPRO-1 uptake in neurons of laminae I-II of spinal cord slices were prevented by the NMDAR antagonist D-AP5, the Src inhibitor PP2 (but not PP3), as well as with the 10Panx and carbenoxolone. Therefore, NMDAR activation in dorsal horn neurons triggers an NMDAR-Src-Panx1 signaling pathway, where Panx1 acts as an enhancing effector in neuropathic pain. This implies that disrupting the NMDAR-Panx1 communication (eg, through Src inhibitors and/or Panx1 blockers) may offer a valuable strategy for managing some forms of chronic pain.

Abstract 4139195: Tipping Point Analysis Identifies C-terminal src kinase-Collagen 22 Regulation by Inflammation in the Pathogenesis of Early-Stage Pulmonary Arterial Hypertension

Inflammatory injury to pulmonary artery endothelial cells (PAECs) is a key pathogenetic event that drives fibrotic vascular remodeling in pulmonary arterial hypertension (PAH), a progressive disease that lacks early stage-specific therapeutic targets. In an inflammatory rodent model of PAH, we established early-stage disease based on abnormalities in right ventricular (RV) afterload, pulmonary vascular resistance, and RV cardiomyocyte phenotype that were evident without substantial elevation in pulmonary artery pressure. We performed RNA-Seq on PAEC mRNA isolated ex vivo from control, early-stage, and advanced-stage PAH rats and deployed a ‘tipping point’ methodology to identify key pulmonary endothelial protein-protein interactions (PPIs) that regulate the transition from control to early-stage PAH in silico . Building on prior data on the intersection between glucose and proline signaling in fibrosis, we interrogated our early-stage PAH network for convergent PPIs involving metabolism and fibrosis endotypes. C-terminal src kinase (Csk), a conserved inhibitor of Src, was predicted to regulate PAEC fibrosis and was increased in early-stage PAH. In human PAECs, inflammatory stimuli affected markers of Src activation as well as collagen 22 (Col22A1). We observed that pulmonary arterial Col22A1 expression increased progressively across all PAH stages in vivo and that arterial Col22A1 expression is upregulated in human PAH. Further, although inflammation upregulated total Csk mRNA and protein expression in HPAECs, it decreased phosphorylation of Src at tyrosine 527 (pTyr527Src), a Csk-dependent brake on Src kinase activity. Targeting Src through small molecule inhibition or adenovirus overexpression of Csk to increase pTyr527Src, in turn, attenuated inflammation-induced Col22A1 in vitro . Taken together, these data suggest that acquired inhibition of Csk is a novel molecular mechanism by which inflammation induces pulmonary endothelial dysfunction, Src activation, and fibrosis. In early-stage PAH, disrupted Csk inhibition of Src may induce transcriptional upregulation of Csk as a negative feedback response to Src activation. These data suggest that the Csk-Src-Col22A1 axis is a novel target for advancing early-stage therapeutics in patients with PAH subtypes characterized by inflammatory vascular injury.

CNSC-53. SHH ACTIVATION IN DEDIFFERENTIATION DURING TUMOR PROGRESSION IN MPNST

Abstract BACKGROUND Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive sarcomas with little progress on outcomes and treatment strategies. Previously, unsupervised analyses of methylome and transcriptome profiles of 108 peripheral nerve sheath tumors uncovered two subgroups of MPNSTs that predict progression-free survival, MPNST-G1 (characterized by SHH-pathway activation) and MPNST-G2 (characterized by WNT/ß-catenin/CCND1-pathway activation). Further, single nuclear RNA sequencing revealed that MPNST-G1 and MPNST-G2 cells resemble neural crest-like and Schwann cell precursor-like cells, respectively. PURPOSE & HYPOTHESIS To examine whether the activation of the SHH pathway in MPNST-G1 cell lines induces overexpression of factors known to play canonical roles in early neural crest cell specification (TWIST1, SNAI2, PAX3, PAX6, SOX9, OTX2) and to investigate the role of Src activation on SHH pathway-induced dedifferentiation. We hypothesize that MPNST-G1 cells will exhibit SHH pathway activation, synergizing with Src to induce the overexpression of early neural crest cell (ENCC) transcription factors. METHODS SHH pathway activation, expression of ENCC transcription factors, and effects of inhibitors were analyzed in MPNST-G1 cells using RT-PCR, western blotting, Alamar Blue assay, Trypan Blue assay, and Soft Agar Transformation assay. RESULTS Compared to MPNST-G2 cells, MPNST-G1 cells displayed SHH-pathway activation, SMO-dependent activation of Src, and elevated expression of the ENCC transcription factors. Sonidegib (SMO inhibitor) and Dasatinib (Src inhibitor) were able to revert dedifferentiation. CONCLUSIONS The SHH-pathway cooperates with Src to promote expression of important transcription factors in dedifferentiation. This finding provides insights into the transformation process of MPNST and novel therapeutic options.

Kinin Receptors B1 and B2 Mediate Breast Cancer Cell Migration and Invasion by Activating the FAK-Src Axis.

Kinin receptors B1 and B2 are involved in migration and invasion in gastric, glioma, and cervical cancer cells, among others. However, the role of kinin receptors in breast cancer cells has been poorly studied. We aimed to reveal the impact of B1 and B2 receptors on migration and invasion in breast cancer cells and demonstrate their capacity to modulate in vivo tumor growth. MDA-MB-231, MCF-7, and T47D cells treated with Lys-des[Arg9]bradykinin (LDBK) or bradykinin (BK) were used to evaluate migration and invasion. Des-[Arg9]-Leu8-BK and HOE-140 were used as antagonists for the B1 and B2 receptors. MDA-MB-231 cells incubated or not with antagonists were subcutaneously inoculated in BALBc NOD/SCID mice to evaluate tumor growth. LDBK and BK treatment significantly increased migration and invasion in breast cancer cells, effects that were negated when antagonists were used. The use of antagonists in vivo inhibited tumor growth. Moreover, the migration and invasion induced by kinins in breast cancer cells were inhibited when focal adhesion kinase (FAK) and Src inhibitors were used. The novelty revealed in our work is that B1 and B2 receptors activated by LDBK and BK induce migration and invasion in breast cancer cells via a mechanism that involves the FAK-Src signaling pathway, and the antagonism of both receptors in vivo impairs breast tumor growth.

SKI-606, a Src inhibitor, ameliorates benzene-induced hematotoxicity via blocking ROS/Src kinase-mediated p38 and Akt signaling pathways

Exposure to benzene causes acute myelosuppression and other hematologic disorders. However, the detailed mechanism by which benzene exerts its severe hematotoxicity and potential treatments still require further deciphering and exploration. Herein, we found that hydroquinone (HQ), a main benzene metabolite, significantly increased intracellular reactive oxygen species (ROS) formation and subsequently caused damage to DNA, leading to impaired colony formation capacity and induction of apoptosis in human hematopoietic stem/progenitor cells (HSPCs) in vitro. The effects were mediated by activation of Src kinase, which subsequently activated the p38 signaling pathway while inhibiting the Akt signaling pathway. The mechanism was further verified by pre-treatment with a Src kinase inhibitor SKI-606, which effectively reversed the dampened self-renewal capacity and increased apoptosis of HSPCs induced by HQ in vitro. Furthermore, administration of SKI-606 partially reversed benzene-induced hematotoxicity and prolonged the survival time in benzene-poisoned mice. Taken together, these findings highlight that HQ-induced hematotoxicity in HSPCs is attributed to the Src kinase-mediated activation of p38 signaling pathway and repression of Akt signaling pathway. Notably, SKI-606 as a tyrosine kinase inhibitor may be a promising and potential agent for alleviating benzene-induced hematotoxicity.

In vitro biomaterial priming of human mesenchymal stromal/stem cells : implication of the Src/JAK/STAT3 pathway in vasculogenic mimicry

Mesenchymal stromal/stem cells (MSC) play a crucial role in promoting neovascularization, which is essential for wound healing. They are commonly utilized as an autologous source of progenitor cells in various stem cell-based therapies. However, incomplete MSC differentiation towards a vascular endothelial cell phenotype questions their involvement in an alternative process to angiogenesis, namely vasculogenic mimicry (VM), and the signal transducing events that regulate their in vitro priming into capillary-like structures. Here, human MSC were primed on top of Cultrex matrix to recapitulate an in vitro phenotype of VM. Total RNA was extracted, and differential gene expression assessed through RNA-Seq analysis and RT-qPCR. Transient gene silencing was achieved using specific siRNA. AG490, Tofacitinib, and PP2 pharmacological effects on VM structures were analyzed using the Wimasis software. In vitro VM occurred within 4 h and was prevented by the JAK/STAT3 inhibitors AG490 and Tofacitinib, as well as by the Src inhibitor PP2. RNA-Seq highlighted STAT3 as a signaling hub contributing to VM when transcripts from capillary-like structures were compared to those from cell monolayers. Concomitant increases in IL6, IL1b, CSF1, CSF2, STAT3, FOXC2, RPSA, FN1, and SNAI1 transcript levels suggest the acquisition of a combined angiogenic, inflammatory and epithelial-to-mesenchymal transition phenotype in VM cultures. Increases in STAT3, FOXC2, RPSA, Fibronectin, and Snail protein expression were confirmed during VM. STAT3 and RPSA gene silencing abrogated in vitro VM. In conclusion, in vitro priming of MSC into VM structures requires Src/JAK/STAT3 signaling. This molecular evidence indicates that a clinically viable MSC-mediated pseudo-vasculature process could temporarily support grafts through VM, allowing time for the host vasculature to infiltrate and remodel the injured tissues.

Exploratory research on the effective chemical basis of tanreqing injection for treating acute lung injury: In vivo, in vitro and in silico

Ethnopharmacological relevanceSepsis-induced acute lung injury (ALI) presents with significant morbidity and mortality in clinical settings. Tanreqing Injection (TRQI) has been clinically recommended for the treatment of ALI; however, the specific active chemical constituents remain unidentified. Aim of the studyThis study aimed to elucidate the potential pharmacologically active components and the underlying mechanisms of TRQI in the treatment of sepsis-induced ALI. Materials and methodsHigh-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) techniques were employed to identify the effective chemical constituents of TRQI. Additionally, an in vitro study was conducted using Raw264.7 macrophage cells stimulated with lipopolysaccharide (LPS) to evaluate the inhibitory effects of TRQI. An acute lung injury model produced by LPS was intraperitoneal injection in mice to assess the ALI-inhibitory effect of TRQI. The lung's pathological characteristics were examined using hematoxylin and eosin staining. Enzyme-linked immunosorbent assay (ELISA) and QPCR were performed to confirm the pharmaceutical effect. Network pharmacology was employed for mechanistic exploration, incorporating GO, and PPI analyses of targets. Src inhibitor and JNK agonist used to investigate the dependence of associated signaling pathways. ResultsCombining pharmacokinetic characteristics, lung first-pass effect and anti-inflammatory effects, the main components of TRQI for treating sepsis induced ALI were narrowed down to seven compounds: chlorogenic acid, scutellarin, wogonoside, oroxyloside, oroxylin A and baicalein. Network pharmacology indicated that Src/JNK signaling pathway, may be the main regulatory pathway for treatment of actue lung injury. Next by using Src inhibitor, Src inhibition partly diminished the protective effects of TRQI in LPS-injected mice. Pretreatment with JNK agonist anisomycin abolished the protective effects of lung injury in vivo. ConclusionsTRQI is injected, the seven compounds could be presented in vivo, which can improve ALI by inhibiting Src-JNK signaling.

Mu-Opioid Receptor (MOR) Dependence of Pain in Chemotherapy-Induced Peripheral Neuropathy.

We recently demonstrated that transient attenuation of Toll-like receptor 4 (TLR4) in dorsal root ganglion (DRG) neurons, can both prevent and reverse pain associated with chemotherapy-induced peripheral neuropathy (CIPN), a severe side effect of cancer chemotherapy, for which treatment options are limited. Given the reduced efficacy of opioid analgesics to treat neuropathic, compared with inflammatory pain, the cross talk between nociceptor TLR4 and mu-opioid receptors (MORs), and that MOR and TLR4 agonists induce hyperalgesic priming (priming), which also occurs in CIPN, we determined, using male rats, whether (1) antisense knockdown of nociceptor MOR attenuates CIPN, (2) and attenuates the priming associated with CIPN, and (3) CIPN also produces opioid-induced hyperalgesia (OIH). We found that intrathecal MOR antisense prevents and reverses hyperalgesia induced by oxaliplatin and paclitaxel, two common clinical chemotherapy agents. Oxaliplatin-induced priming was also markedly attenuated by MOR antisense. Additionally, intradermal morphine, at a dose that does not affect nociceptive threshold in controls, exacerbates mechanical hyperalgesia (OIH) in rats with CIPN, suggesting the presence of OIH. This OIH associated with CIPN is inhibited by interventions that reverse Type II priming [the combination of an inhibitor of Src and mitogen-activated protein kinase (MAPK)], an MOR antagonist, as well as a TLR4 antagonist. Our findings support a role of nociceptor MOR in oxaliplatin-induced pain and priming. We propose that priming and OIH are central to the symptom burden in CIPN, contributing to its chronicity and the limited efficacy of opioid analgesics to treat neuropathic pain.

C-Src Is Responsible for Mitochondria-Mediated Arrhythmic Risk in Ischemic Cardiomyopathy.

Increased mitochondrial Ca2+ uptake has been implicated in the QT prolongation and lethal arrhythmias associated with nonischemic cardiomyopathy. We attempted to define the role of mitochondria in ischemic arrhythmic risk and to identify upstream regulators. Myocardial infarction (MI) was induced in wild-type FVB/NJ mice by ligation of the left anterior descending coronary artery. Western blot, immunoprecipitation, ECG telemetry, and patch-clamp techniques were used. After MI, c-Src (proto-oncogene tyrosine-protein kinase Src) and its active form (p-Src Y416) were increased. The activation of c-Src was associated with increased diastolic Ca2+ sparks, action potential duration prolongation, and arrhythmia in MI mice. c-Src upregulation and arrhythmia could be reversed by treatment of mice with the Src inhibitor PP1 but not with the inactive analogue PP3. Tyrosine phosphorylated mitochondrial Ca2+ uniporter (MCU) was upregulated in the heart tissues of MI mice and patients with ischemic cardiomyopathy. In a heterologous expression system, c-Src could bind MCU and phosphorylate MCU tyrosines. Overexpression of wild-type c-Src significantly increased the mitochondrial Ca2+ transient while overexpression of dominant-negative c-Src significantly decreased the mitochondrial Ca2+ transient. c-Src inhibition by PP1, MCU inhibition by Ru360, or MCU knockdown could reduce the action potential duration, Ca2+ sparks, and arrhythmia after MI. The human heart tissue showed that patients with ischemic cardiomyopathy had significantly increased c-Src active form associated with increased MCU tyrosine phosphorylation and ventricular arrhythmia. MI leads to increased c-Src active form that results in MCU tyrosine phosphorylation, increased mitochondrial Ca2+ uptake, QT prolongation, and arrhythmia, suggesting c-Src or MCU may represent novel antiarrhythmic targets.

Bioinformatics and computational studies of chabamide F and chabamide G for breast cancer and their probable mechanisms of action

Globally, the prevalence of breast cancer (BC) is increasing at an alarming level, despite early detection and technological improvements. Alkaloids are diverse chemical groups, and many within this class have been reported as potential anticancer compounds. Chabamide F (F) and chabamide G (G) are two dimeric amide alkaloids found in a traditional medicinal plant, Piper chaba, and possess significant cytotoxic effects. However, their scientific rationalization in BC remains unknown. Here, we aimed to investigate their potential and molecular mechanisms for BC through in silico approaches. From network pharmacology, we identified 64 BC-related genes as targets. GO and KEGG studies showed that they were involved in various biological processes and mostly expressed in BC-related pathways such as RAS, PI3K-AKT, estrogen, MAPK, and FoxO pathways. However, PPI analysis revealed SRC and AKT1 as hub genes, which play key roles in BC tumorigenesis and metastasis. Molecular docking revealed the strong binding affinity of F (− 10.7 kcal/mol) and G (− 9.4 and − 11.7 kcal/mol) for SRC and AKT1, respectively, as well as the acquisition of vital residues to inhibit them. Their long-term stability was evaluated using 200 ns molecular dynamics simulation. The RMSD, RMSF, Rg, and SASA analyses showed that the G-SRC and G-AKT1 complexes were excellently stable compared to the control, dasatinib, and capivasertib, respectively. Additionally, the PCA and DCCM analyses revealed a significant reduction in the residual correlation and motions. By contrast, the stability of the F-SRC complex was greater than that of the control, whereas it was moderately stable in complex with AKT1. The MMPBSA analysis demonstrated higher binding energies for both compounds than the controls. In particular, the binding energy of G for SRC and AKT1 was − 120.671 ± 16.997 and − 130.437 ± 19.111 kJ/mol, respectively, which was approximately twice as high as the control molecules. Van der Waal and polar solvation energies significantly contributed to this energy. Furthermore, both of them exhibited significant interactions with the binding site residues of both proteins. In summary, this study indicates that these two molecules could be a potential ATP-competitive inhibitor of SRC and an allosteric inhibitor of AKT1.

Neighboring Effect-Initiated Supramolecular Nanocomplex with Sequential Infiltration as Irreversible Apoptosis Inducer for Synergetic Chemo-Immunotherapy.

Chemotherapy-based combination regimens are recommended as first-line treatment for colorectal cancer. However, multidrug resistance (MDR) and limited drug infiltration in tumor microenvironment remain critical challenges. Herein, a pH/redox dual activated supramolecular DAS@CD-OxPt (IV) nanoparticles (NPs) via host-guest molecular recognition to achieve relay drugs delivery of active oxaliplatin (OxPt (IV)) and Src inhibitor dasatinib (DAS) between tumor cells is developed. DAS@CD-OxPt (IV) NPs exhibit prolonged circulation in the blood and intra-tumoral retention. Triggered by the endo/lysosome (pH 5.0), flexible DAS@CD-OxPt (IV) NPs exhibited proton-driven in situ assembly to form nanofiber in tumor cells. Dual chemotherapeutic agents released from DAS@CD-OxPt (IV) NPs synergistically cause irreversible DNA damage by blocking p53-mediated DNA repair. Supramolecular nanofibers can further serve as the "ammunition depot" to continuously release drugs from dying cells and transport them into neighboring tumor cells, leading to domino-like cell death and enhanced immunogenicity. Furthermore, DAS@CD-OxPt (IV) NPs combined with immune checkpoint blockade (ICB) therapy strikingly suppress CT26 tumor growth and pulmonary metastasis.

Targeting the tyrosine kinase Src in endothelium attenuates inflammation and atherogenesis induced by disturbed flow.

Previous studies have shown that Src can regulate inflammation and tumour progression. However, the mechanisms by which Src regulates the inflammatory response of vascular endothelium and atherogenesis are currently poorly understood. This study aimed to investigate the role of Src in endothelial inflammation and atherogenesis, as well as the underlying mechanisms. Real-time quantitative PCR was used to measure the mRNA levels of inflammatory genes. The phosphorylation and localization of proteins were examined using western blotting and immunofluorescence, respectively. The level of p-Src Y416 in mouse endothelium was directly determined using en face staining. Endothelial-specific knockdown of Src was achieved by tail vein injection of AAV-sgSrc in ApoE-/-; Cas9LSL/LSL; Cdh5-cre mice. Atherosclerosis was induced by partial ligation of the carotid artery. Oscillatory shear stress (OSS) promotes the phosphorylation of Src at Y416 in endothelial cells, and Piezo1 is required for this regulatory process. Overexpression of constitutively active Src promotes endothelial inflammation, as well as phosphorylation of Stat3 (at Y705) and its nuclear translocation. Endothelial inflammation induced by OSS was abolished by the Src inhibitor dasatinib or si-Src. Dasatinib, when administered orally, reduced endothelial inflammation and plaque formation in ApoE-/- mice induced by partial carotid artery ligation. Additionally, plaque formation was decreased in the ligated left carotid artery of mice with endothelial-specific Src knockdown. Disturbed flow promotes endothelial inflammation and atherogenesis through the Piezo1-Src-Stat3 pathway. Therefore, inhibiting Src in endothelial cells could be a promising therapeutic strategy to treat atherogenesis.