PI3K Signaling Pathway Research Articles

Abstract LB_A24: Baicalein suppresses tumorigenesis of the ER𝛼 positive of breast cancer cells by targeting multiple signaling pathways

Abstract The anti-tumor properties of baicalein, a flavonoid compound, have been extensively documented. However, the specific effects of baicalein on ER𝛼 positive breast cancer cell lines, namely T47-D and MCF-7, are less well-known. In this study, we conducted comprehensive in vitro experiments to elucidate the comparative impacts of baicalein on these two cell lines. Our results demonstrate that baicalein exerts robust anti-cancer effects, inducing morphological changes, reducing colony formation, and inhibiting cell proliferation in both T47-D and MCF-7 cells. These effects were more pronounced at higher concentrations. Additionally, baicalein treatment impeded the migratory capabilities of both cell lines, as shown by wound healing assays. Furthermore, our data show that baicalein triggers apoptosis, leading to an increase in early apoptotic, late apoptotic, and dead cells. This apoptotic process appears to be caspase 3/7-dependent. Interestingly, the pro-apoptotic effects were more potent in T47-D cells, accompanied by significant upregulation of pro-apoptotic genes and downregulation of anti-apoptotic markers. Baicalein treatment also induced oxidative stress and DNA damage, evidenced by an increase in reactive oxygen species (ROS) and phosphorylation of ATM and H2A.X. Importantly, baicalein inhibited the PI3K signaling pathway, a key mediator of cancer progression. Genome-wide transcriptomic analysis revealed significant changes in gene expression patterns, with baicalein treatment causing both upregulation and downregulation of several genes. Pathway analysis revealed that cell cycle and ferroptosis pathways were activated in both cell lines upon baicalein treatment. In conclusion, our findings highlight the potential of baicalein as a therapeutic agent against breast cancer, while also revealing nuanced differences in response between the T47-D and MCF-7 breast cancer cell lines. Citation Format: Besa Xhabija, Aidan J. McLaughlin, Ibiere Lovelyn Epelle, Tristan H. Nielson, Elisabeta Ujeniuc, Mariam I. Duhaini. Baicalein suppresses tumorigenesis of the ER𝛼 positive of breast cancer cells by targeting multiple signaling pathways [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A24.

Oncogenetic-Driven Targeted Therapy for Relapsed/Refractory T-Cell Acute Lymphoblastic Leukemia : A French ALL-Target Observatory Report

Introduction T-cell acute lymphoblastic leukemia (T-ALL) is an orphan disease diagnosed mostly in adolescent and young adults. In adult population, 5-10% of T-ALL patients (pts) will be primary refractory and 30-40% will relapse. In relapse/refractory (R/R) patients, standard of care treatments, including nelarabine, yield response rate of about 20-40% and responses are of short duration. On behalf of the GRAALL (Group for Research on Adult Acute Lymphoblastic Leukemia), we launched the ALL-TARGET project combining a precision medicine platform dedicated to R/R T-ALL and T-cell lymphoblastic lymphoma (T-LL), and an observatory to evaluate therapeutic proposals based on mutational profile and intracellular signaling pathways alterations. Objectives and methods Leukemic samples from pts with R/R T-ALL/LL were shipped to the GRAALL T-ALL central laboratory in France (V. Asnafi, Hôpital Necker). Biological characterization comprised oncogenetic, phenotypic and in some cases functional analysis. Clinical data from R/R T-ALL/LL pts were collected in a real-life observatory (NCT05832125). Adults pts were eligible if an oncogenetic characterization was available at diagnosis or relapse, and if they received a salvage, either with conventional therapy or with a targeted therapeutic option (TTO). For example, TTOs included Tofacitinib and Venetoclax (Tofa/Ven) in case of IL7R (CD127) expression or IL7R-pathway alterations (IL7R ALT), 5-azacytidine and Venetoclax (Aza/Ven) in case of T-ALL/LL with epigenetic regulators alterations (DNMT3A, ASXL1, PHF6, TET2, PRC2, IDH1/2, SRSF2...) or Temsirolimus, Erwinase and Venetoclax (Tem/Erw/Ven) in case of PI3K signaling pathway alterations (PI3K ALT). The ALL-TARGET Observatory primary endpoint was overall response rate (ORR) including complete remission (CR), CR with incomplete hematological recovery and partial response. We report here the results of the first pts included, with a focus on those who received TTOs as salvage therapy. Results Eighty-nine were analyzed, including 80 T-ALL and 9 T-LL. Sex ratio was 3 and median age at diagnosis was 37.5y, and 51y at the time of the first TTO. Seventy-one pts were in relapse (79.7%) and 18 primary refractory (20.2%). Relapses occurred after a median first CR duration of 16.6 months (range: 2.5-92), with 62.8% in bone marrow, 30% in CNS and 30.3% in other extramedullary sites. Phenotype at diagnosis was available for 68 pts, including 36 Early T-cell Precursor (ETP or near-ETP) (52.9%), 6 immature non-ETP (8.8%), 20 cortical (29.4%) and 6 mature phenotype (8.8%). IL7R-expression was available for 53 pts, of whom 40 were IL7R+ (75.5%) and 13 IL7R- (24.5%). Out of 50 samples with available BCL2-expression, 47 were BCL2+ (94%) and 3 BCL2- (6%). IL7R ALT were evidenced in 41 pts (48.2%), PI3K ALT in 21 pts (24.7%), RAS pathway alterations (RAS ALT) in 19 pts (22.4%) and epigenetic dysregulation in 42 pts (49.4%). Ten pts harbored TP53/ATM mutations (11.7%) . These different alterations could coexist. At relapse, 33 phenotypes were reported, showing 22 ETP (66.6%), 2 immature non-ETP (6%) and 9 cortical (27.3%). IL7R-expression was available for 26 pts, among which 21 were IL7R+ (63.6%) and 5 IL7R- (19.2%). NGS was performed in 24 patients, revealing IL7R ALT in 9 pts (37.5%), PI3K ALT in 6 pts (25%), RAS ALT in 5 pts (20.8%) and epigenetic dysregulation in 15 pts (62.5%). Nine patients had TP53 mutations (37.5%). Twenty-five patients received a TTO, including 14 Aza/Ven (56%), 8 Tofa/Ven (32%), 2 Tem/Erw/Ven (8%) and 1 Trametinib/Ven (4%). Among these, 8 were in first salvage (32%), 10 in 2 nd (40%), and 6 in 3 rd/4 th salvage (28%). Of note, 14 patients received Ven associated with chemotherapy, including 4 Nelarabine-Ven, and 3 Ven in monotherapy. By 3 months, the cumulative incidence of response under the chosen TTO was 70.7% (95%CI:51-88) (Figure). Ten of 14 patients were in response after Aza/Ven (ORR 71.4%), 4 of 8 patients after Tofa/Ven (50%) and 2 of 2 patients treated with Tem/Erw/Ven (100%). Five patients were bridged to allogeneic stem cell transplantation. Conclusion Our results demonstrate the feasibility of the ALL-TARGET project. A better knowledge of the oncogenetic landscape of T-ALL, and a close collaboration between clinicians and biologists, resulted in individualized treatment strategies. With a 3 months cumulative incidence of response of 70%, TTOs appear to be a promising approach in R/R T-ALL.

The role and mechanisms of PD-L1 in immune evasion during Talaromyces marneffei infection

Talaromycosis, caused by Talaromyces marneffei (T. marneffei), is a systemic fungal disease that involves dissemination throughout the body. The ability of T. marneffei to evade the immune system is considered a crucial factor in its persistent infection, although the specific mechanisms are not yet fully understood. This study aims to investigate the molecular mechanisms underlying the occurrence of latent T. marneffei infection and immune evasion. The gene expression profile analysis in T. marneffei-infected mouse revealed that Pd-l1 exhibited the highest correlation strength with other hub genes, with a median of 0.60 (IQR: 0.50–0.69). T. marneffei infection upregulated the expression of PD-1 and PD-L1 in PBMCs from HIV patients, which was also observed in the T. marneffei-infected mouse and macrophage models. Treatment with a PD-L1 inhibitor significantly reduced fungal burden in the liver and spleen tissues of infected mice and in the kupffer-CTLL-2 co-culture system. PD-L1 inhibitor treatment increased CTLL-2 cell proliferation and downregulated the expression of PD-1, SHP-2, and p-SHP-2, indicating the activation of T cell viability and T cell receptor signaling pathway. Additionally, treatment with a PI3K inhibitor downregulated PD-L1 in T. marneffei-infected kupffer cells. Similar results were observed with treatment using the T. marneffei cell wall virulence factor β-glucan. Overall, T. marneffei infection upregulated PD-L1 expression in HIV / T. marneffei patients, mice, and kupffer cells. Treatment with a PD-L1 inhibitor significantly reduced fungal burden, while activating T cell activity and proliferation, thereby promoting fungal clearance. Furthermore, the PI3K signaling pathway may be involved in the regulation of PD-L1 by T. marneffei.

The p110α/ΔNp63α complex mutations in triple-negative breast cancer: Potential targets for transcriptional-based therapies.

Hotspot mutations occurring in the p110α domain of the PIK3CA gene, specifically p110αH1047R/L increase tumor metastasis and cell motility in triple-negative breast cancer (TNBC). These mutations also affect the transcriptional regulation of ΔNp63α, a significant isoform of the p53 protein involved in cancer progression. This study attempts to investigate the transcriptional impact of p110αH1047R/L mutations on the PIK3CA/ΔNp63α complex in TNBC carcinogenesis. We performed site-directed mutagenesis to introduce p110αH1047R/L mutations and evaluated their oncogenic effects on the growth, invasion, migration, and apoptosis of three different TNBC cell lines in vitro. We investigated the impact of these mutations on the p110α/ΔNp63α complex and downstream transcriptional signaling pathways at the gene and protein levels. Additionally, we used bioinformatics techniques such as molecular dynamics simulations and protein-protein docking to gain insight into the stability and structural changes induced by the p110αH1047R/L mutations in the p110α/ΔNp63α complex and downstream signaling pathway. The presence of PIK3CA oncogenic hotspot mutations in the p110α/ΔNp63α complex led to increased scattering of TNBC cells during growth, migration, and invasion. Our in vitro mutagenesis assay showed that the p110αH1047R/L mutations activated the PI3K-Akt-mTOR and tyrosine kinase receptor pathways, resulting in increased cell proliferation, invasion, and apoptosis in TNBC cells. These mutations decreased the repressing effect of ΔNp63α on the p110α kinase domain, leading to the enhancement of downstream signaling pathways of PI3K and tyrosine kinase receptors and oncogenic transformation in TNBC. Additionally, our findings suggest that the physical interaction between the DNA binding domain of ΔNp63α and the kinase domain of p110α may be partially impaired, potentially leading to alterations in the conformation of the p110α/ΔNp63α complex. Our findings suggest that targeting the p110αH1047R/L mutations in TNBC could be a promising strategy for developing transcriptional-based therapies. Restoring the interaction between ΔNp63α and the p110α kinase domain, which is disrupted by these mutations, may provide a new approach to treating TNBC.

Abstract 17407: A Small-molecule Screening Strategy to Promote Cardiomyocyte Proliferation That Protects the Heart From Ischemic Injury

Restoring the ability of adult cardiac myocytes (ACMs) to proliferate and promote heart regeneration have been proposed as a promising therapeutic strategy. We have demonstrated that selective depletion of H3K9me3 by lysine demethylase 4D (KDM4D) overexpression increases ACM cell cycle activity, but proliferation was limited. To improve this, we performed a small-molecule screening in combination with KDM4D on neonatal rat cardiomyocytes (NRVMs), using a library of kinase inhibitors targeting different signaling pathways. Our study revealed that compounds targeting the PI3K and JAK signaling pathways significantly potentiated the effects of KDM4D and promote NRVM proliferation in vitro. Specifically, a JAK inhibitor (JAKi2) in combination with Ad-KDM4D treatment resulted in approximately 100%, 34%, and 62% increase in EdU+, phospho-H3+, and total cell number compared to control, respectively. To validate these findings in vivo, we tested JAKi2 in an inducible KDM4D transgenic mouse model (iKDM4D). The animals underwent ischemia/reperfusion (I/R) to induce infarction, and JAKi2 was administrated orally for up to 8 weeks. We found both early and late effects. 48 hours after I/R, the infarct size in iKDM4D+JAKi2 hearts was approximately 4-fold smaller than that in vehicle-treated or JAKi2 alone groups, and the combination increased EdU+, Ki67+, and phospho-H3+ CM number separately by 3.2-fold, 2.9-fold, and 6.5-fold. At 2 weeks post-I/R, the scar size was smaller in iKDM4D+JAKi2 hearts compared to vehicle-treated mice (9.0% vs 1.8%, P <0.05), and the combination resulted in a 3-fold, 5.5-fold, and 8.5-fold increases in EdU+, Ki67+, and phospho-H3+ CMs, respectively. Moreover, the iKDM4D+JAKi2 hearts demonstrated progressive improvement in left ventricular ejection fraction (26.8%, 27.3%, and 25.5%) and fraction shorten (37.9%, 38.1%, and 35.0%) at 2 weeks, 4 weeks, and 8 weeks post-I/R. The increased estimated ACM number and mononucleated CMs at 8 weeks post-I/R support that CM division was increased in the iKDM4D+JAKi2 hearts. These findings suggest that a combination therapy involving JAKi2, along with KDM4D, promotes robust CM proliferation and protects the heart after ischemic injury.

IGF2BP1 Stabilizes and Inhibits Degradation of Pro-Oncogenic Transcripts in ETV6-RUNX1 Positive B-Cell Acute Lymphoblastic Leukemia

Insulin like growth factor 2 mRNA binding protein 1(IGF2BP1) belongs to a family of oncofetal proteins expressed in embryonic life followed by re-emergence during malignant transformation. It is an RNA binding protein known to be dysregulated and overexpressed in multiple malignancies. We have demonstrated its specific overexpression in ETV6-RUNX1 translocated B-cell Acute Lymphoblastic Leukemia (B-ALL). RNA immunoprecipitation using anti-IGF2BP1 antibody, followed by sequencing (RIP-Seq) in Reh cell line (ETV6-RUNX1 positive) identified multiple oncogenic transcripts belonging to the non-canonical NF-κB and PI3K signaling pathways. Overexpression of genes from these pathways (IL6ST, GADD45A, NFAT5, MDM2, CDK6, FGFR1) was also observed in our B-ALL patient samples specifically in the ETV6-RUNX1 positive patients (Total n=111; ETV6-RUNX1 positive: 39) in comparison to MACS sorted, peripheral blood CD19 positive B-cells from healthy volunteers as well as in comparison to non ETV6-RUNX1 translocated B-Acute lymphoblastic leukemia patients. This was also corroborated by public B-ALL gene expression data from the TARGET database in the cBioportal. However, knockout of IGF2BP1 in Reh cell line using CRISPR-Cas9 technology led to significant downregulation of only some of these targets. This implied that the expression of some of the mRNA targets binding to IGF2BP1 was not influenced by its knockout. To better elucidate the role played by IGF2BP1 in the stability of its binding mRNA targets, we decided to study the rate of degradation of target transcripts. mRNA stability was assessed to quantify the rate of degradation of target mRNA after inhibition of transcription using actinomycin. Since actinomycin causes global transcriptional block, we first quantified the effect of actinomycin on the expression of multiple (n=7) housekeeping genes against each other, and selected 3 ( HGPRT, B2M and PPIA) which showed least change after actinomycin treatment. These 3 housekeeping genes were used for further quantification of target transcripts. Dual specificity phosphatase 1( DUSP1) a transcript known to be rapidly degraded, was used to validate the transcriptional inhibition by actinomycin. We quantified the rate of degradation of target transcripts in Reh cells using BTYNB, a small molecule inhibitor of IGF2BP1. BTYNB binds to IGF2BP1 and prevents binding to target transcripts which leads to their destabilization and reduced half-life. We used a combination of BTYNB and actinomycin to study rate of degradation after IGF2BP1 inhibition. Effect of IGF2BP1 on the stability of three target mRNAs involved in non-canonical NF-κB signalling pathway was quantified by comparing their rate of degradation in BTYNB treated cells against the DMSO (vehicle) treated cells. Rate of degradation was measured by actinomycin treatment of both these groups followed by hourly RNA isolation and qPCR over 12 hours. MYC is a known mRNA binding target of IGF2BP1. BTYNB was also shown to decrease the expression of MYC mRNA by accelerating its degradation. Simultaneously there was an increase in the expression of MYC pre-mRNA indicating a possible for IGF2BP1 in splicing of MYC RNA. In the NF-κB signalling pathway, we had previously identified that NFAT5 expression was not altered significantly after IGF2BP1 knockout even though it binds to IGF2BP1. BTYNB followed by actinomycin treatment demonstrated a dramatic increase in the rate of degradation of NFAT5 implying that IGF2BP1 prevents the degradation of NFAT5 transcript and directly influences its stability. Interestingly, the rates of degradation of other targets like IL6ST and GADD45 were not altered. These findings suggest a heterogeneous influence of IGF2BP1 on the stability of its target transcripts. Some of the transcripts seem to be directly stabilised by IGF2BP1 while others may be influenced indirectly. Taken together, these findings suggest a role of IGF2BP1 in multiple phases of the RNA synthesis and degradation warranting further studies to elucidate role of IGF2BP1 in each of these steps. IGF2BP1 appears to have a critical role in preventing the degradation and stabilizing MYC and NFAT5 transcripts.

Active compounds from Calendula officinalis flowers act via PI3K and ERK signaling pathways to offer neuroprotective effects against Parkinson's disease.

Calendula officinalis flowers, associated with diverse biological effects, could be utilized as functional food ingredients to play a crucial role in human health. In this study, we examined the anti-PD activity of C. officinalis flower extracts and investigated their bioactive compounds and molecular mechanisms based on LC-MS/MS assay, bioinformatic exploration and invitro treatment of SH-SY5Y cells. C. officinalis extracts exhibited significant positive effects on the length and fluorescence density of the dopaminergic neuron region in zebrafish larvae. At 10 μg/mL, the extract restored the length to 96.54% and fluorescence density to 87.77% of the control values, which was equivalent to the effect of a positive drug, indicating the extract's powerful potential to alleviate PD symptoms. Five active compounds, including chlorogenic acid, 3,4-dicaffeoylquinic acid (DA), rutin, isorhamnetin 3-O-glucoside (IG) and calenduloside E (CE) were identified in extracts by LC-QTOF-MS/MS. Hsp90α, PI3K and ERK were revealed as core targets of DA, IG and CE in relation to anti-PD activity. The compounds docked deeply within the pocket region of Hsp90α protein, and their binding energies (∆G b) were -6.93 kcal/mol (DA), -6.51 kcal/mol (IG) and -3.03 kcal/mol (CE), respectively. Subsequently, they concurrently activated the PI3K/Akt signaling pathway and inhibited the ERK signaling pathway, thereby preventing neuronal death and alleviating neuronal degeneration. These compounds from C. officinalis could be potent nutraceutical agents with protective properties that may shield dopaminergic neurons against the damage caused by PD. Our findings provide a basis for utilizing the C. officinalis flowers in functional foods.

Smart-seq2 Technology Reveals a Novel Mechanism That Zearalenone Inhibits the In Vitro Maturation of Ovine Oocytes by Influencing TNFAIP6 Expression.

Zearalenone (ZEN), a non-steroidal estrogenic fungal toxin widely present in forage, food, and their ingredients, poses a serious threat to animal and human reproductive health. ZEN also threatens ovine, a major source of human food and breeding stock. However, the mechanisms underlying the impact of ZEN on the in vitro maturation (IVM) of ovine oocytes remain unclear. This study aimed to elucidate these mechanisms using the Smart-seq2 technology. A total of 146 differentially expressed genes were obtained, using Smart-seq2, from sheep oocytes cultured in vitro after ZEN treatment. ZEN treatment inhibited RUNX2 and SPP1 expression in the PI3K signaling pathway, leading to the downregulation of THBS1 and ultimately the downregulation of TNFAIP6; ZEN can also decrease TNFAIP6 by reducing PTPRC and ITGAM. Both inhibit in vitro maturation of ovine oocytes and proliferation of cumulus cells by downregulating TNFAIP6. These findings provide data and a theoretical basis for elucidating ZEN's toxicity mechanisms, screening therapeutic drugs, and reducing ZEN-related losses in the ovine industry.

Evaluation of the anti-inflammatory effects of PI3Kδ/γ inhibitors for treating acute lung injury

Phosphatidylinositol 3-kinase delta (PI3Kδ) and gamma (PI3Kγ) are predominantly located in immune and hematopoietic cells. It is well-established that PI3Kδ/γ plays important roles in the immune system and participates in inflammation; hence, it could be a potential target for anti-inflammatory therapy. Currently, several PI3K inhibitors are used clinically to treat cancers with aberrant PI3K signaling; however, their role in treating acute respiratory inflammatory diseases has rarely been explored. Herein, we investigated the potential anti-inflammatory activities of several pharmacological PI3K inhibitors, including marketed drugs idelalisib (PI3Kδ), duvelisib (PI3Kδ/γ), and copanlisib (pan-PI3K with preferential α/δ) and the clinical drug eganelisib (PI3Kγ), for treating acute lung injury (ALI). In the lipopolysaccharide-induced RAW264.7 macrophage inflammatory model, the four inhibitors significantly suppressed proinflammatory cytokine expression by inhibiting the PI3K signaling pathway. Oral administration of PI3K inhibitors markedly improved lung injury in a murine model of ALI. PI3K pathway inhibition decreased inflammatory cell infiltration and totalprotein levels, as well as reduced the expression of associated lung inflammatory factors. Collectively, all four representative PI3K inhibitors exerted prominent anti-inflammatory properties, indicating that PI3K δ and/or γ inhibition could be ideal targets to treat respiratory inflammatory diseases by reducing the inflammatory response. The findings of the current study provide a new basis for utilizing PI3K inhibitors to treat acute respiratory inflammatory diseases.

The IGF1 Signaling Pathway: From Basic Concepts to Therapeutic Opportunities.

Insulin-like growth factor 1 (IGF1) is a peptide growth factor with important functions in multiple aspects of growth, development and metabolism. The biological actions of IGF1 are mediated by the IGF1 receptor (IGF1R), a cell-surface protein that is evolutionarily related to the insulin receptor (InsR). The effects of IGF1 are moderated by a group of binding proteins (IGFBPs) that bind and transport the ligand in the circulation and extracellular fluids. In mechanistic terms, IGF1R function is linked to the MAPK and PI3K signaling pathways. Furthermore, IGF1R has been shown to migrate to cell nucleus, where it functions as a transcriptional activator. The co-localization of IGF1R and MAPK in the nucleus is of major interest as it suggests novel mechanistic paradigms for the IGF1R-MAPK network. Given its potent anti-apoptotic and pro-survival roles, and in view of its almost universal pattern of expression in most types of cancer, IGF1R has emerged as a promising molecular target in oncology. The present review article provides a concise overview of key scientific developments in the research area of IGF and highlights a number of more recent findings, including its nuclear migration and its interaction with oncogenes and tumor suppressors.

The correlation between clinical outcomes and genomic analysis with high risk factors for the progression of osteosarcoma.

Osteosarcoma (OS) is a rare but aggressive malignancy. Despite previous reports, molecular characterization of this disease is not well understood, and little is known regarding OS in Chinese patients. Herein, we analyzed the genomic signatures of 73 Chinese OS cases. TP53, NCOR1, LRP1B, ATRX, RB1, and TFE3 were the most frequently mutated gene in our OS cohort. In addition, the genomic analysis of Western OS patients was performed. Notably, there were remarkable disparities in mutational landscape, base substitution pattern, and tumor mutational burden between the Chinese and Western OS cohorts. Specific molecular mechanisms, including DNA damage repair (DDR) gene mutations, copy number variation (CNV) presence, aneuploidy, and intratumoral heterogeneity, were associated with disease progression. Additionally, 30.1% of OS patients carried clinically actionable alterations, which were mainly enriched in PI3K, MAPK, DDR, and RTK signaling pathways. A specific molecular subtype incorporating DDR alterations and CNVs was significantly correlated with distant metastasis-free survival and event-free survival, and this correlation was observed in all subgroups with different characteristics. These findings comprehensively elucidated the genomic profile and revealed novel prognostic factors in OS, which would contribute to understanding this disease and promoting precision medicine of this population.

Myeloid-derived suppressor cells and tolerogenic dendritic cells are distinctively induced by PI3K and Wnt signaling pathways

Imbalanced immune responses are a prominent hallmark of cancer and autoimmunity. Myeloid cells can be overly suppressive, inhibiting protective immune responses or inactive not controlling autoreactive immune cells. Understanding the mechanisms that induce suppressive myeloid cells, such as myeloid-derived suppressor cells (MDSCs) and tolerogenic dendritic cells (TolDCs), can facilitate the development of immune-restoring therapeutic approaches. MDSCs are a major barrier for effective cancer immunotherapy by suppressing antitumor immune responses in cancer patients. TolDCs are administered to patients to promote immune tolerance with the intent to control autoimmune disease. Here, we investigated the development and suppressive/tolerogenic activity of human MDSCs and TolDCs to gain insight into signaling pathways that drive immunosuppression in these different myeloid subsets. Moreover, monocyte-derived MDSCs (M-MDSCs) generated invitro were compared to M-MDSCs isolated from head-and-neck squamous cell carcinoma patients. PI3K-AKT signaling was identified as being crucial for the induction of human M-MDSCs. PI3K inhibition prevented the downregulation of HLA-DR and the upregulation of reactive oxygen species and MerTK. In addition, we show that the suppressive activity of dexamethasone-induced TolDCs is induced by β-catenin-dependent Wnt signaling. The identification of PI3K-AKT and Wnt signal transduction pathways as respective inducers of the immunomodulatory capacity of M-MDSCs and TolDCs provides opportunities to overcome suppressive myeloid cells in cancer patients and optimize therapeutic application of TolDCs. Lastly, the observed similarities between generated- and patient-derived M-MDSCs support the use of invitro-generated M-MDSCs as powerful model to investigate the functionality of human MDSCs.

Cholinergic signaling of muscarinic receptors directly involves in the neuroprotection of muscone by inducing Ca2+ antagonism and maintaining mitochondrial function

Ethnopharmacological relevanceMusk, a traditional Chinese medicine, is broadly used in inducing resuscitation and refreshing the mind, activating blood and alleviating pain. It is commonly used for the treatment of ischemic stroke, and muscone is its core medicinal component. Aim of the studyThe aim of this study was to explore whether muscone ameliorates neuronal damage through cholinergic signaling of muscarinic receptors. Materials and methodsThe effects of muscone were tested in a rat model of middle cerebral artery occlusion (MCAO) as well as injured neurons induced by oxygen-glucose deprivation (OGD) in PC12 cells. Cell counting kit 8 (CCK8) assay was used to measure the cell viability, and the production of lactate dehydrogenase (LDH) and adenosine-triphosphate (ATP) were examined by kit. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), tetramethylrhodamine ethyl ester (TMRE) and Fluo-4 acetoxymethyl ester (Fluo-4 AM) staining were used to demonstrate effect of muscone on the reactive oxygen species (ROS) level, mitochondria membrane potential (MMP) and intracellular Ca2+ measurement in cells respectively, in which all of those staining was visualized by laser confocal microscope. For in vivo experiments, rats' cerebral blood flow was measured using laser Doppler blood flowmetry to evaluate the MCAO model, and a modified neurological severity score (mNSS) was used to assess the recovery of neurological function. Calculate infarct rate was measured by 2,3,5-Triphenyl Tetrazolium Chloride (TTC) staining. Except DCFH-DA and Fluo-4 AM staining, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolylcarbocyanine iodide (JC-1) staining was used to observe intracellular Ca2+ measurement in brain cells. Protein levels in cells and tissues were detected by Western blot. ResultsPretreatment with muscone significantly improved the cell viability, lactic acid production, mitochondrial membrane potential collapse and function, Ca2+ overload, ROS generation, and cell apoptosis in OGD PC12 cells. Muscone also regulated PI3K, ERK and AKT signal pathways by activating cholinergic signaling of muscarinic receptors in PC12 cells induced with OGD. More importantly, the blocking of cholinergic signaling of muscarinic receptors by atropine significantly reduces the neuroprotective effects of muscone, including the cell viability, Ca2+ efflux, and mitochondrial repair. Furthermore, muscone was found to effectively alleviate mitochondrial dysfunction and elevated levels of ROS induced by the MCAO in the brain tissue. Notably, this beneficial effect of muscone was attenuated by atropine but not by (+)-Sparteine. ConclusionsOur study indicates that muscone exerts its neuroprotective effects by activating muscarinic receptors of cholinergic signaling, thus providing a promising therapeutic target for the treatment of OGD-induced nerve injury in stroke. The findings suggest that these treatments may hold potential benefits for stroke patients.

CCL2 Knockdown Attenuates Inflammatory Response After Spinal Cord Injury Through the PI3K/Akt Signaling Pathway: Bioinformatics Analysis and Experimental Validation.

Spinal cord injury (SCI) is a common clinical problem in orthopedics with a lack of effective treatments and drug targets. In the present study, we performed bioinformatic analysis of SCI datasets GSE464 and GSE45006 in the Gene Expression Omnibus (GEO) public database and experimentally validated CCL2 expression in an animal model of SCI. This was followed by stimulation of PC-12 cells using hydrogen peroxide to construct a cellular model of SCI. CCL2 expression was knocked down using small interfering RNA (si-CCL2), and PI3K signaling pathway inhibitors and activators were used to validate and observe the changes in downstream inflammation. Through data mining, we found that the inflammatory chemokine CCL2 and PI3K/Akt signaling pathways after SCI expression were significantly increased, and after peroxide stimulation of PC-12 cells with CCL2 knockdown, their downstream cellular inflammatory factor levels were decreased. The PI3K/Akt signaling pathway was blocked by PI3K inhibitors, and the downstream inflammatory response was suppressed. In contrast, when PI3K activators were used, the inflammatory response was enhanced, indicating that the CCL2-PI3K/Akt signaling pathway plays a key role in the regulation of the inflammatory response. This study revealed that the inflammatory chemokine CCL2 can regulate the inflammatory response of PC-12 cells through the PI3K/Akt signaling pathway, and blocking the expression of the inflammatory chemokine CCL2 may be a promising strategy for the treatment of secondary injury after SCI.

Impact of human ovarian tissue manipulation on follicles: evidence of a potential first wave of follicle activation during fertility preservation procedures.

The aim of this study was to investigate the impact of processing human ovarian tissue on follicle activation dynamics. Fresh ovarian tissue was retrieved from 9 women undergoing laparoscopic surgery for benign conditions. Biopsies from each patient were divided into 3 fragments, the first of which was immediately fixed in the operating room (T0) and the second and third just after processing at 25 (T25) and (T90) 90 min. To evaluate follicle activation, markers of the PI3K and Hippo signaling pathways were immunolabeled at each time point, targeting phospho-Akt (p-Akt) by immunohistochemistry and yes-associated protein (YAP) cellular localization in the granulosa cell layer by immunofluorescence. Four hundred forty primordial follicles were evaluated for p-Akt and 420 for YAP. Significantly stronger p-Akt expression was observed at T25 (23.01 ± 13.45%; p=0.04) and T90 (38.99 ± 25.21%; p<0.001) than at T0 (2.72 ± 3.35%). A significant nucleus-to-cytoplasm shift in YAP was detected at T25 (1.21 ± 0.25; p=0.015 compared to T0 (0.95 ± 0.09), while T90 (1.10 ± 0.16) values were similar to T25. Our data prove that ovarian tissue manipulation significantly impacts follicle dynamics by stimulating the PI3K and Hippo signaling pathways involved in primordial follicle activation. Further experimental evidence must nevertheless be gathered to understand and gain control of follicle activation mechanisms in non-physiological conditions (like ovarian tissue manipulation), in order to optimize fertility preservation and restoration strategies.

Ferrostatin-1 post-treatment attenuates acute kidney injury in mice by inhibiting ferritin production and regulating iron uptake-related proteins.

Acute kidney injury (AKI) is a common and serious medical condition with high morbidity and mortality. Recent research has highlighted ferroptosis, a novel form of programmed cell death, as a potential therapeutic target in mitigating renal tubular injury in AKI. Ferrostatin-1, a specific ferroptosis inhibitor, has been demonstrated to prevent renal injury through ferroptosis inhibition. Utilizing a murine AKI model, we investigated the effects of Ferrostatin-1 by administering it post-injury. Through high-throughput sequencing and pathological analysis, we focused on the critical role of ferroptosis-related pathways in the treatment. Ferrostatin-1 post-conditioning effectively mitigated oxidative damage and reduced iron content associated with AKI. Additionally, critical ferroptosis-related proteins, such as GPX4, SLC7A11, NRF2, and FTH1, exhibited increased expression levels. In vitro, Ferrostatin-1 treatment of HK-2 cells significantly diminished lipid peroxidation and iron accumulation. Furthermore, Ferrostatin-1 was found to downregulate the PI3K signalling pathway. Ferrostatin-1 acted as a potential ferroptosis inhibitor with the capacity to enhance antioxidant defences. This study suggests that Ferrostatin-1 could serve as a promising novel strategy for improving the treatment of AKI and promoting recovery from the condition.

Chitosan oligosaccharide suppresses osteosarcoma malignancy by inhibiting CEMIP via the PI3K/AKT/mTOR pathway

Osteosarcoma is a malignant bone tumor that is prone to metastasize early and primarily affects children and adolescents. Cell migration-inducing protein (CEMIP) plays a crucial role in the progression and malignancy of various tumor diseases, including osteosarcoma. Chitosan oligosaccharide (COS), an oligomer isolated from chitin, has been found to have significant anti-tumor activity in various cancers. This study investigates the effects of COS on CEMIP expression in osteosarcoma and explores the underlying mechanism. In present study, in vitro experiments were conducted to confirm the inhibitory activity of COS on human osteosarcoma cells. Our results demonstrate that COS possesses inhibitory effects against human osteosarcoma cells and significantly suppresses CEMIP expression in vitro. Next, we studied the inhibition of the expression of CEMIP by COS and then performed bioinformatics analysis to explore the potential inhibitory mechanism of COS against signaling pathways involved in regulating CEMIP expression. Bioinformatics analysis predicted a close association between the PI3K signaling pathway and CEMIP expression and that the inhibitory effect of COS on CEMIP expression may be related to PI3K signaling pathway regulation. The results of this study show that COS treatment significantly inhibits CEMIP expression and the PI3K/AKT/mTOR signaling pathway, as observed both in vitro and in vivo. This study demonstrates that COS could inhibit the expression of CEMIP, which is closely related to osteosarcoma malignancy. This inhibitory effect may be attributed to the inhibition of the PI3K/AKT/mTOR signaling pathway in vitro and in vivo.

Transcriptome Studies Reveal the N6-Methyladenosine Differences in Testis of Yaks at Juvenile and Sexual Maturity Stages.

Studying the mechanism of spermatogenesis is key to exploring the reproductive characteristics of male yaks. Although N6-methyladenosine (m6A) RNA modification has been reported to regulate spermatogenesis and reproductive function in mammals, the molecular mechanism of m6A in yak testis development and spermatogenesis remains largely unknown. Therefore, we collected testicular tissue from juvenile and adult yaks and found that the m6A level significantly increased after sexual maturity in yaks. In MeRIP-seq, 1702 hypermethylated peaks and 724 hypomethylated peaks were identified. The hypermethylated differentially methylated RNAs (DMRs) (CIB2, AK1, FOXJ2, PKDREJ, SLC9A3, and TOPAZ1) mainly regulated spermatogenesis. Functional enrichment analysis showed that DMRs were significantly enriched in the adherens junction, gap junction, and Wnt, PI3K, and mTOR signaling pathways, regulating cell development, spermatogenesis, and testicular endocrine function. The functional analysis of differentially expressed genes showed that they were involved in the biological processes of mitosis, meiosis, and flagellated sperm motility during the sexual maturity of yak testis. We also screened the key regulatory factors of testis development and spermatogenesis by combined analysis, which included BRCA1, CREBBP, STAT3, and SMAD4. This study indexed the m6A characteristics of yak testicles at different developmental stages, providing basic data for further research of m6A modification regulating yak testicular development.

SNORC knockdown alleviates inflammation, autophagy defect and matrix degradation of chondrocytes in osteoarthritis development.

Excessive inflammation and autophagy defect of chondrocytes play important roles in the pathological process of osteoarthritis (OA). The present study aimed to clarify the roles of small novel rich in cartilage (SNORC) in these pathological changes of chondrocytes in OA. Bioinformatics analysis of GEO dataset GSE207881 displayed that SNORC was a potential biomarker for OA. As confirmed by quantitative real-time PCR, immunohistochemical staining and western blotting, SNORC was significantly up-regulated in cartilage of OA rat model and interleukin (IL)-1β-stimulated primary rat articular chondrocytes in contrast to their corresponding normal control. Knocking down SNORC in IL-1β-induced chondrocytes obviously suppressed the production of nitric oxide (NO), IL-6, tumor necrosis factor (TNF)-α and prostaglandin E2 (PGE2) to alleviate inflammation, and reduced the protein levels of a disintegrin and metalloproteinase with thrombospondin 5 (ADAMTS5) and matrix metallopeptidase (MMP)13 and elevated collagen type 2 alpha 1 (COL2A1) level to improve matrix degradation. Down-regulation of SNORC increased Beclin1 expression and LC3II/LC3I ratio, but suppressed p62 expression to restore impaired autophagy in IL-1β-induced chondrocytes. Moreover, down-regulating SNORC mitigated mitochondrial dysfunction and apoptosis in IL-1β-stimulated chondrocytes. Mechanically, SNORC simultaneously activated the phosphatidylinositol-3-kinase/serine threonine kinase (PI3K/AKT) and c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway in the IL-1β-induced chondrocyte, while re-activating the PI3K and JNK signals abolished the suppressive effect of down-regulating SNORC on IL-1β-induced chondrocyte damage. In a word, SNORC knockdown alleviates inflammation, matrix degradation, autophagy defect and excessive apoptosis of chondrocytes during OA development via suppressing the PI3K and JNK signaling pathway.

Paroxetine's effect on the proinflammatory cytokine stimulation and intracellular signaling pathways in J774.2 cells.

Paroxetine is extensively utilized in the management of depressive and anxious conditions. Paroxetine works by increasing serotonin levels in nerve cells in the brain. However, limited information is available regarding the direct effects of paroxetine on macrophage cells. Macrophages are a type of leukocytes involved in the body's immune response, playing a crucial role in combating infections. The impact of paroxetine on macrophages has been explored in research, although a comprehensive understanding is still pending. This study aimed to research the potential of administering paroxetine to J774.2 macrophage cells to stimulate the release of GM-CSF, TNF-α, IL-12p40, and IL-6 cytokines. Additionally, we examined the mechanisms of action of paroxetine on the p38 signaling pathway, which is involved in cytokine production, and the PI3K pathway, which is an important mechanism in intracellular signaling. Our findings revealed that paroxetine induced an inflammatory response in macrophages by promoting cytokine synthesis in a non-lipopolysaccharide (LPS) environment. We observed that paroxetine triggered the inflammatory response through the PI3K signaling pathway while suppressing the p38 signaling pathway.