ROCK1 regulates glycolysis in pancreatic cancer via the c-MYC/PFKFB3 pathway.

BACKGROUND: Dysregulation of Rho-associated coiled coil-containing protein kinases (ROCKs) is involved in the metastasis and progression of various malignant tumors. However, how one of the isomers, ROCK1, regulates glycolysis in tumor cells is incompletely understood. Here, we attempted to elucidate how ROCK1 influences pancreatic cancer (PC) progression by regulating glycolytic activity. METHODS: The biological function of ROCK1 was analyzed in vitro by establishing a silenced cell model. Coimmunoprecipitation confirmed the direct binding between ROCK1 and c-MYC, and a luciferase reporter assay revealed the binding of c-MYC to the promoter of the PFKFB3 gene. These results were verified in animal experiments. RESULTS: ROCK1 was highly expressed in PC tissues and enriched in the cytoplasm, and its high expression was associated with a poor prognosis. Silencing ROCK1 inhibited the proliferation and migration of PC cells and promoted their apoptosis. Mechanistically, ROCK1 directly interacted with c-MYC, promoted its phosphorylation (Ser 62) and suppressed its degradation, thereby increasing the transcription of the key glycolysis regulatory factor PFKFB3, enhancing glycolytic activity and promoting PC growth. Silencing ROCK1 increased gemcitabine (GEM) sensitivity in vivo and in vitro. CONCLUSIONS: ROCK1 promotes glycolytic activity in PC cells and promotes PC tumor growth through the c-MYC/PFKFB3 signaling pathway. ROCK1 knockdown can inhibit PC tumor growth in vivo and increase the GEM sensitivity of PC tumors, providing a crucial clinical therapeutic strategy for PC.

Staphylococcus aureus Conquers Host by Hijacking Mitochondria via PFKFB3 in Epithelial Cells.

Staphylococcus aureus (S. aureus) persists within mammary epithelial cells for an extended duration, exploiting the host metabolic resources to facilitate replication. This study revealed a mechanism by which intracellular S. aureus reprograms host metabolism, with PFKFB3 playing a crucial role in this process. Mechanistically, S. aureus induced mitochondrial damage, leading to increased levels of mitochondrial reactive oxygen species (mROS) and dysfunction in electron transport chain (ETC). Moreover, S. aureus shifted the balance of mitochondrial dynamics from fusion to fission, subsequently activating PINK1-PRKN-dependent mitophagy, causing loss of the sirtuin 3 (SIRT3) to stabilize hypoxic inducible factor 1α (HIF1α), and shifting the host metabolism toward enhanced glycolysis. The inhibition of PFKFB3 reversed the mitochondrial damage and degradation of SIRT3 induced by S. aureus. Overall, our findings elucidate the mechanism by which S. aureus reprograms host metabolism and offer insights into the treatment of S. aureus infection.

Targeting the reprogrammed metabolism in H3.3K27M pediatric high-grade gliomas.

Recent studies in Diffuse Midline Gliomas (DMG) demonstrated a strong connection between epigenome dysregulation and metabolic rewiring. Here, we evaluated the value of targeting a glycolytic protein named Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3 (PFKFB3) in H3.3K27M DMG. We observed that the viability of H3.3K27M cells is dramatically reduced by PFK15, a potent inhibitor of PFKFB3. Furthermore, PFKFB3 inhibition induced apoptosis and G2/M arrest. Interestingly, CRISPR-Knockout of the K27M mutant allele has a synergistic effect on the observed phenotype. Altogether, we identified PFKFB3 as a new target for H3.3K27M DMG, making PFK15 a potential candidate for future animal studies and clinical trials.

MiR-106a-5p targets PFKFB3 and improves sepsis through regulating macrophage pyroptosis and inflammatory response.

The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) is a critical regulator of glycolysis and plays a key role in modulating the inflammatory response, thereby contributing to the development of inflammatory diseases such as sepsis. Despite its importance, the development of strategies to target PFKFB3 in the context of sepsis remains challenging. In this study, we employed a miRNA-based approach to decrease PFKFB3 expression. Through multiple meta-analyses, we observed a downregulation of miR-106a-5p expression and an upregulation of PFKFB3 expression in clinical sepsis samples. These changes were also confirmed in blood monocytes from patients with early sepsis and from a mouse model of lipopolysaccharide (LPS)-induced sepsis. Overexpression of miR-106a-5p significantly decreased the LPS-induced increase in glycolytic capacity, inflammatory response, and pyroptosis in macrophages. Mechanistically, we identified PFKFB3 as a direct target protein of miR-106a-5p and demonstrated its essential role in LPS-induced pyroptosis and inflammatory response in macrophages. Furthermore, treatment with agomir-miR-106a-5p conferred a protective effect in an LPS mouse model of sepsis, but this effect was attenuated in myeloid-specific Pfkfb3 KO mice. These findings indicate that miR-106a-5p inhibits macrophage pyroptosis and inflammatory response in sepsis by regulating PFKFB3-mediated glucose metabolism, representing a potential therapeutic option for the treatment of sepsis.

The glycolytic enzyme PFKFB3 drives kidney fibrosis through promoting histone lactylation-mediated NF-κB family activation.

Persistently elevated glycolysis in kidney has been demonstrated to promote chronic kidney disease (CKD). However, the underlying mechanism remains largely unclear. Here, we observed that 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a key glycolytic enzyme, was remarkably induced in kidney proximal tubular cells (PTCs) following ischemia-reperfusion injury (IRI) in mice, as well as in multiple etiologies of patients with CKD. PFKFB3 expression was positively correlated with the severity of kidney fibrosis. Moreover, patients with CKD and mice exhibited increased urinary lactate/creatine levels and kidney lactate, respectively. PTC-specific deletion of PFKFB3 significantly reduced kidney lactate levels, mitigated inflammation and fibrosis, and preserved kidney function in the IRI mouse model. Similar protective effects were observed in mice with heterozygous deficiency of PFKFB3 or those treated with a PFKFB3 inhibitor. Mechanistically, lactate derived from PFKFB3-mediated tubular glycolytic reprogramming markedly enhanced histone lactylation, particularly H4K12la, which was enriched at the promoter of NF-κB signaling genes like Ikbkb, Rela, and Relb, activating their transcription and facilitating the inflammatory response. Further, PTC-specific deletion of PFKFB3 inhibited the activation of IKKß, I κ B α, and p65 in the IRI kidneys. Moreover, increased H4K12la levels were positively correlated with kidney inflammation and fibrosis in patients with CKD. These findings suggest that tubular PFKFB3 may play a dual role in enhancing NF-κB signaling by promoting both H4K12la-mediated gene transcription and its activation. Thus, targeting the PFKFB3-mediated NF-κB signaling pathway in kidney tubular cells could be a novel strategy for CKD therapy.

The predictive value of PFKFB3 in bladder cancer prognosis.

6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 (PFKFB3) influences cancer progression via participating in tumor aerobic glycolysis. In this study, we aimed to evaluate the prognostic significance of PFKFB3 in bladder cancer (BLCA) patients by analyzing a combination of publicly available databases, clinical patient data, and bladder tumor samples from our hospital. Single-cell and bulk RNA-seq data of bladder cancer, obtained from ENA, GEO, and TCGA databases, were utilized for our analysis. The results indicated that PFKFB3 mRNA expression was markedly elevated in bladder cancer compared to paired normal tissue. Furthermore, BLCA patients with high PFKFB3 expression exhibited a significantly worse prognosis (P < 0.05). To validate these findings, clinical data and immunohistochemistry staining were performed on specimens obtained from 89 BLCA patients who underwent radical cystectomy at either Qingdao University Affiliated Hospital or Peking Union Medical College Hospital. The findings from this verification process confirmed that high expression of PFKFB3 serves as a biomarker for predicting worse prognosis in BLCA patients (OR: 2.462, 95 % CI: 1.202-5.042, P = 0.012). To facilitate clinical application, we developed a nomogram based on four variables, including PFKFB3 expression, to predict the survival of BLCA patients. Importantly, this nomogram demonstrated a low mean prediction error of 0.03. Taken together, our findings suggest that PFKFB3 has the potential to serve as both a prognostic biomarker and a therapeutic target for BLCA patients.

PFKFB3 regulates breast cancer tumorigenesis and Fulvestrant sensitivity by affecting ERα stability.

Estrogen receptor alpha (ERα) is expressed in approximately 70% of breast cancer cases and determines the sensitivity and effectiveness of endocrine therapy. 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase3 (PFKFB3) is a glycolytic enzyme that is highly expressed in a great many human tumors, and recent studies have shown that it plays a significant role in improving drug sensitivity. However, the role of PFKFB3 in regulating ERα expression and the underlying mechanism remains unclear. Here, we find by using immunohistochemistry (IHC) that PFKFB3 is elevated in ER-positive breast cancer and high expression of PFKFB3 resulted in a worse prognosis. In vitro and in vivo experiments verify that PFKFB3 promotes ER-positive breast cancer cell proliferation. The overexpression of PFKFB3 promotes the estrogen-independent ER-positive breast cancer growth. In an estrogen-free condition, RNA-sequencing data from MCF7 cells treated with siPFKFB3 showed enrichment of the estrogen signaling pathway, and a luciferase assay demonstrated that knockdown of PFKFB3 inhibited the ERα transcriptional activity. Mechanistically, down-regulation of PFKFB3 promotes STUB1 binding to ERα, which accelerates ERα degradation by K48-based ubiquitin linkage. Finally, growth of ER-positive breast cancer cells in vivo was more potently inhibited by fulvestrant combined with the PFKFB3 inhibitor PFK158 than for each drug alone. In conclusion, these data suggest that PFKFB3 is identified as an adverse prognosis factor for ER-positive breast cancer and plays a previously unrecognized role in the regulation of ERα stability and activity. Our results further explores an effective approach to improve fulvestrant sensitivity through the early combination with a PFKFB3 inhibitor.

Endothelial nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome regulation in atherosclerosis.

AIMS: The activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in endothelial cells (ECs) contributes to vascular inflammation in atherosclerosis. Considering the high glycolytic rate of ECs, we delineated whether and how glycolysis determines endothelial NLRP3 inflammasome activation in atherosclerosis. METHODS AND RESULTS: Our results demonstrated a significant up-regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a key regulator of glycolysis, in human and mouse atherosclerotic endothelium, which positively correlated with NLRP3 levels. Atherosclerotic stimuli up-regulated endothelial PFKFB3 expression via sterol regulatory element-binding protein 2 (SREBP2) transactivation. EC-selective haplodeficiency of Pfkfb3 in Apoe-/- mice resulted in reduced endothelial NLRP3 inflammasome activation and attenuation of atherogenesis. Mechanistic investigations revealed that PFKFB3-driven glycolysis increased the NADH content and induced oligomerization of C-terminal binding protein 1 (CtBP1), an NADH-sensitive transcriptional co-repressor. The monomer form, but not the oligomer form, of CtBP1 was found to associate with the transcriptional repressor Forkhead box P1 (FOXP1) and acted as a transrepressor of inflammasome components, including NLRP3, caspase-1, and interleukin-1ß (IL-1ß). Interfering with NADH-induced CtBP1 oligomerization restored its binding to FOXP1 and inhibited the glycolysis-dependent up-regulation of NLRP3, Caspase-1, and IL-1ß. Additionally, EC-specific overexpression of NADH-insensitive CtBP1 alleviates atherosclerosis. CONCLUSION: Our findings highlight the existence of a glycolysis-dependent NADH/CtBP/FOXP1-transrepression pathway that regulates endothelial NLRP3 inflammasome activation in atherogenesis. This pathway represents a potential target for selective PFKFB3 inhibitors or strategies aimed at disrupting CtBP1 oligomerization to modulate atherosclerosis.

PFKFB3-mediated glycolysis in hepatic stellate cells promotes liver regeneration.

Hepatic stellate cells (HSCs) perform a significant function in liver regeneration (LR) by becoming active. We propose to investigate if activated HSCs enhance glycolysis via PFKFB3, an essential glycolytic regulator, and whether targeting this pathway could be beneficial for LR. The liver and isolated HSCs of mice subjected to 2/3 partial hepatectomy (PHx) exhibited a significant rise in PFKFB3 expression, as indicated by quantitative RT-PCR analyses and Western blotting. Also, the primary HSCs of mice subjected to PHx have a significant elevation of the glycolysis level. Knocking down PFKFB3 significantly diminished the enhancement of glycolysis by PDGF in human LX2 cells. The hepatocyte proliferation in mice treated with PHx was almost completely prevented when the PFKFB3 inhibitor 3PO was administered, emerging that PFKFB3 is essential in LR. Furthermore, there was a decline in mRNA expression of immediate early genes and proinflammatory cytokines. In terms of mechanism, both the p38 MAP kinase and ERK1/2 phosphorylation in LO2 cells and LO2 proliferation were significantly reduced by the conditioned medium (CM) obtained from LX2 cells with either PFKFB3 knockdown or inhibition. Compared to the control group, isolated hepatocytes from 3PO-treated mice showed decreased p38 MAP kinase and ERK1/2 phosphorylation and proliferation. Thus, LR after PHx involves the activation of PFKFB3 in HSCs, which enhances glycolysis and promotes lactate production, thereby facilitating hepatocyte proliferation via the p38/ERK MAPK signaling pathway.

Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis.

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

PFKFB3 facilitates cell proliferation and migration in anaplastic thyroid carcinoma via the WNT/ß-catenin signaling pathway.

PURPOSE: Despite the involvement of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase3 (PFKFB3) in the proliferation and metastasis of diverse tumor types, its biological functions and related molecular mechanisms in anaplastic thyroid carcinoma (ATC) remain largely unclear. METHODS: Datasets from the Gene Expression Omnibus, the Cancer Genome Atlas and immunohistochemistry (IHC) analyses were employed to measure the expression level of PFKFB3 in ATC. A series of assays were performed to analyze the role of PFKFB3 and its inhibitor KAN0438757 in ATC cell proliferation and migration. Furthermore, Western blotting (WB), IHC and luciferase reporter assay were conducted to investigate the potential mechanisms underlying the involvement of PFKFB3 and KAN0438757 in ATC. Additionally, we established a subcutaneous xenograft tumor model in nude mice to evaluate the in vivo tumor growth. RESULTS: PFKFB3 exhibited a significant increase in its expression level in ATC tissues. The overexpression of PFKFB3 resulted in the stimulation of ATC cell proliferation and migration. Furthermore, this overexpression was associated with the elevated expression levels of p-AKT (ser473), p-GSK3α/ß (ser21/9), nuclear ß-catenin, fibronectin1 (FN1), matrix metallopeptidase 9 (MMP-9) and cyclin D1. It also promoted the nuclear translocation of ß-catenin and the transcription of downstream molecules. Conversely, contrasting results were observed with the downregulation or KAN0438757-mediated inhibition of PFKFB3 in ATC cells. The selective AKT inhibitor MK2206 was noted to reverse the increased expression of p-AKT (ser473) and p-GSK3α/ß (ser21/9) induced by PFKFB3 overexpression. The level of lactate was increased in PFKFB3-overexpressing ATC cells, while the presence of KAN0438757 inhibited lactate production. Moreover, the simultaneous use of PFKFB3 downregulation and KAN0438757 was found to suppress subcutaneous tumor growth in vivo. CONCLUSION: PFKFB3 can enhance ATC cell proliferation and migration via the WNT/ß-catenin signaling pathway and plays a crucial role in the regulation of aerobic glycolysis in ATC cells.

PFKFB3 in neovascular eye disease: unraveling mechanisms and exploring therapeutic strategies.

BACKGROUND: Neovascular eye disease is characterized by pathological neovascularization, with clinical manifestations such as intraocular exudation, bleeding, and scar formation, ultimately leading to blindness in millions of individuals worldwide. Pathologic ocular angiogenesis often occurs in common fundus diseases including proliferative diabetic retinopathy (PDR), age-related macular degeneration (AMD), and retinopathy of prematurity (ROP). Anti-vascular endothelial growth factor (VEGF) targets the core pathology of ocular angiogenesis. MAIN BODY: In recent years, therapies targeting metabolism to prevent angiogenesis have also rapidly developed, offering assistance to patients with a poor prognosis while receiving anti-VEGF therapy and reducing the side effects associated with long-term VEGF usage. Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key enzyme in targeted metabolism, has been shown to have great potential, with antiangiogenic effects and multiple protective effects in the treatment of neovascular eye disease. In this review, we summarize the mechanisms of common types of neovascular eye diseases; discuss the protective effect and potential mechanism of targeting PFKFB3, including the related inhibitors of PFKFB3; and look forward to the future exploration directions and therapeutic prospects of PFKFB3 in neovascular eye disease. CONCLUSION: Neovascular eye disease, the most common and severely debilitating retinal disease, is largely incurable, necessitating the exploration of new treatment methods. PFKFB3 has been shown to possess various potential protective mechanisms in treating neovascular eye disease. With the development of several drugs targeting PFKFB3 and their gradual entry into clinical research, targeting PFKFB3-mediated glycolysis has emerged as a promising therapeutic approach for the future of neovascular eye disease.

Drug-repurposing by virtual and experimental screening of PFKFB3 inhibitors for pancreatic cancer therapy.

Pancreatic cancer (PC) is the most frequently occurring cancer, with few effective treatments and a 5-year survival rate of only about 11%. It is characterized by stiff interstitium and pressure on blood vessels, leading to an increased glycolytic metabolism. PFKFB3 plays an important role in glycolysis, and its products (fructose-2,6-bisphosphate), which are allosteric PFK1 activators, limit the glycolytic rate. In this study, 14 PFKFB3 inhibitors were obtained by virtually screening the FDA-approved compound library. Subsequently, the in-vitro investigations confirmed that Lomitapide and Cabozantinib S-malate exhibit the excellent potential to inhibit PFKFB3. The combined administration of Lomitapide and Gemcitabine at a certain molar ratio indicated an enhanced anti-tumor effect in Orthotopic Pancreatic Cancer (OPC) models. This investigation provides a new treatment strategy for PC therapy.

RORα inhibits gastric cancer proliferation through attenuating G6PD and PFKFB3 induced glycolytic activity.

BACKGROUND: Glycolysis is critical for harvesting abundant energy to maintain the tumor microenvironment in malignant tumors. Retinoic acid-related orphan receptor α (RORα) has been identified as a circadian gene. However, the association of glycolysis with RORα in regulating gastric cancer (GC) proliferation remains poorly understood. METHODS: Bioinformatic analysis and retrospective study were utilized to explore the role of RORα in cell cycle and glycolysis in GC. The mechanisms were performed in vitro and in vivo including colony formation, Cell Counting Kit-8 (CCK-8), Epithelial- mesenchymal transition (EMT) and subcutaneous tumors of mice model assays. The key drives between RORα and glycolysis were verified through western blot and chip assays. Moreover, we constructed models of high proliferation and high glucose environments to verify a negative feedback and chemoresistance through a series of functional experiments in vitro and in vivo. RESULTS: RORα was found to be involved in the cell cycle and glycolysis through a gene set enrichment analysis (GSEA) algorithm. GC patients with low RORα expression were not only associated with high circulating tumor cells (CTC) and high vascular endothelial growth factor (VEGF) levels. However, it also presented a positive correlation with the standard uptake value (SUV) level. Moreover, the SUVmax levels showed a positive linear relation with CTC and VEGF levels. In addition, RORα expression levels were associated with glucose 6 phosphate dehydrogenase (G6PD) and phosphofructokinase-2/fructose-2,6-bisphosphatase (PFKFB3) expression levels, and GC patients with low RORα and high G6PD or low RORα and high PFKFB3 expression patterns had poorest disease-free survival (DFS). Functionally, RORα deletion promoted GC proliferation and drove glycolysis in vitro and in vivo. These phenomena were reversed by the RORα activator SR1078. Moreover, RORα deletion promoted GC proliferation through attenuating G6PD and PFKFB3 induced glycolytic activity in vitro and in vivo. Mechanistically, RORα was recruited to the G6PD and PFKFB3 promoters to modulate their transcription. Next, high proliferation and high glucose inhibited RORα expression, which indicated that negative feedback exists in GC. Moreover, RORα deletion improved fluorouracil chemoresistance through inhibition of glucose uptake. CONCLUSION: RORα might be a novel biomarker and therapeutic target for GC through attenuating glycolysis.

Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis.

BACKGROUND: Hepatocellular carcinoma (HCC) remains a leading life-threatening health challenge worldwide, with pressing needs for novel therapeutic strategies. Sphingosine kinase 1 (SphK1), a well-established pro-cancer enzyme, is aberrantly overexpressed in a multitude of malignancies, including HCC. Our previous research has shown that genetic ablation of Sphk1 mitigates HCC progression in mice. Therefore, the development of PF-543, a highly selective SphK1 inhibitor, opens a new avenue for HCC treatment. However, the anti-cancer efficacy of PF-543 has not yet been investigated in primary cancer models in vivo, thereby limiting its further translation. METHODS: Building upon the identification of the active form of SphK1 as a viable therapeutic target in human HCC specimens, we assessed the capacity of PF-543 in suppressing tumor progression using a diethylnitrosamine-induced mouse model of primary HCC. We further delineated its underlying mechanisms in both HCC and endothelial cells. Key findings were validated in Sphk1 knockout mice and lentiviral-mediated SphK1 knockdown cells. RESULTS: SphK1 activity was found to be elevated in human HCC tissues. Administration of PF-543 effectively abrogated hepatic SphK1 activity and significantly suppressed HCC progression in diethylnitrosamine-treated mice. The primary mechanism of action was through the inhibition of tumor neovascularization, as PF-543 disrupted endothelial cell angiogenesis even in a pro-angiogenic milieu. Mechanistically, PF-543 induced proteasomal degradation of the critical glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, thus restricting the energy supply essential for tumor angiogenesis. These effects of PF-543 could be reversed upon S1P supplementation in an S1P receptor-dependent manner. CONCLUSIONS: This study provides the first in vivo evidence supporting the potential of PF-543 as an effective anti-HCC agent. It also uncovers previously undescribed links between the pro-cancer, pro-angiogenic and pro-glycolytic roles of the SphK1/S1P/S1P receptor axis. Importantly, unlike conventional anti-HCC drugs that target individual pro-angiogenic drivers, PF-543 impairs the PFKFB3-dictated glycolytic energy engine that fuels tumor angiogenesis, representing a novel and potentially safer therapeutic strategy for HCC.

Ginsenoside Rg3 overcomes tamoxifen resistance through inhibiting glycolysis in breast cancer cells.

Tamoxifen (TAM) resistance poses a significant clinical challenge in human breast cancer and exhibits high heterogeneity among different patients. Rg3, an original ginsenoside known to inhibit tumor growth, has shown potential for enhancing TAM sensitivity in breast cancer cells. However, the specific role and underlying mechanisms of Rg3 in this context remain unclear. Aerobic glycolysis, a metabolic process, has been implicated in chemotherapeutic resistance. In this study, we demonstrate that elevated glycolysis plays a central role in TAM resistance and can be effectively targeted and overcome by Rg3. Mechanistically, we observed upregulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key mediator of glycolysis, in TAM-resistant MCF-7/TamR and T-47D/TamR cells. Crucially, PFKFB3 is indispensable for the synergistic effect of TAM and Rg3 combination therapy, which suppresses cell proliferation and glycolysis in MCF-7/TamR and T-47D/TamR cells, both in vitro and in vivo. Moreover, overexpression of PFKFB3 in MCF-7 cells mimicked the TAM resistance phenotype. Importantly, combination treatment significantly reduced TAM-resistant MCF-7 cell proliferation in an in vivo model. In conclusion, this study highlights the contribution of Rg3 in enhancing the therapeutic efficacy of TAM in breast cancer, and suggests that targeting TAM-resistant PFKFB3 overexpression may represent a promising strategy to improve the response to combination therapy in breast cancer.

Brassica oleracea L. extract ameliorates isoproterenol-induced myocardial injury by regulating HIF-1α-mediated glycolysis.

Brassica oleracea L. (BO) is an important vegetable with proven health benefits. This study aimed to elucidate the constituents of BO leaf extract (BOE) and evaluate its effect on myocardial injury. For this purpose, the constituents of BOE were identified using ultra-high performance liquid chromatography with quadrupole time-of- flight mass spectrometry, and 26 compounds were determined, including glucosinolates, sulfur compounds, alkaloids, phenolic acids, flavones, and two other kinds of compounds. The effects of BOE on myocardial cells were evaluated using isoproterenol (ISO)-treated H9C2 cells and Wistar rats, and the results revealed that BOE could inhibit cardiomyocyte hypertrophy and reduce the levels of B-type natriuretic peptide, nitric oxide, reactive oxygen species, lactic acid, and pyruvic acid. Meanwhile, BOE could increase the levels of mitochondrial membrane potential. Moreover, BOE could reduce the levels of apoptosis- and glycolysis-related proteins. Taken together, our data demonstrated that BOE treatment could alleviate ISO-induced myocardial cell injury by downregulating apoptosis and glycolysis signals.

PFKFB3-Meditated Glycolysis via the Reactive Oxygen Species-Hypoxic Inducible Factor 1α Axis Contributes to Inflammation and Proliferation of Staphylococcus aureus in Epithelial Cells.

Mastitis caused by antibiotic-resistant strains of Staphylococcus aureus is a significant concern in the livestock industry due to the economic losses it incurs. Regulating immunometabolism has emerged as a promising approach for preventing bacterial inflammation. To investigate the possibility of alleviating inflammation caused by S aureus infection by regulating host glycolysis, we subjected the murine mammary epithelial cell line (EpH4-Ev) to S aureus challenge. Our study revealed that S aureus can colonize EpH4-Ev cells and promote inflammation through hypoxic inducible factor 1α (HIF1α)-driven glycolysis. Notably, the activation of HIF1α was found to be dependent on the production of reactive oxygen species (ROS). By inhibiting PFKFB3, a key regulator in the host glycolytic pathway, we successfully modulated HIF1α-triggered metabolic reprogramming by reducing ROS production in S aureus-induced mastitis. Our findings suggest that there is a high potential for the development of novel anti-inflammatory therapies that safely inhibit the glycolytic rate-limiting enzyme PFKFB3.

PFKFB3 promotes endometriosis cell proliferation via enhancing the protein stability of ß-catenin.

Endometriosis is a common inflammatory disease in women of reproductive age and is highly associated with infertility. However, the molecular mechanism of endometriosis remains unclear. 6-Phosphofructose-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) is a key enzyme in glycolysis and plays an important regulatory role in the development of cancer. Here we found that PFKFB3 is highly expressed in endometriotic tissues. PFKFB3 promotes the proliferation and growth of endometriosis cells. Meanwhile, PFKFB3 promotes glycolysis in endometriosis cells. Furthermore, PFKFB3 promotes migration and invasion of endometriosis cells. On this basis, we found that PFKFB3 promotes epithelial-mesenchymal transition (EMT) in endometriosis cells. PFKFB3 interacts with the essential factor of EMT, ß-catenin, and promotes the protein stability of ß-catenin. In addition, the PFKFB3 inhibitor PFK-015 inhibites the growth of endometriosis cells and the development of endometrial tissue. In conclusion, our study shows that PFKFB3 plays an important role in the development of endometriosis and provides new ideas for the clinical diagnosis or treatment of endometriosis.

Targeted metabolomics reveals PFKFB3 as a key target for elemene-mediated inhibition of glycolysis in prostate cancer cells.

BACKGROUND: Elemene, an active anticancer extract derived from Curcuma wenyujin, has well-documented anticarcinogenic properties. Nevertheless, the role of elemene in prostate cancer (PCa) and its underlying molecular mechanism remain elusive. PURPOSE: This study focuses on investigating the anti-PCa effects of elemene and its underlying mechanisms. METHODS: Cell-based assays, including CCK-8, scratch, colony formation, cell cycle, and apoptosis experiments, to comprehensively assess the impact of elemene on PCa cells (LNCaP and PC3) in vitro. Additionally, we used a xenograft model with PC3 cells in nude mice to evaluate elemene in vivo efficacy. Targeted metabolomics analysis via HILIC-MS/MS was performed to investigate elemene potential target pathways, validated through molecular biology experiments, including western blotting and gene manipulation studies. RESULTS: In this study, we discovered that elemene has remarkable anti-PCa activity in both in vitro and in vivo settings, comparable to clinical chemotherapeutic drugs but with fewer side effects. Using our established targeted metabolomics approach, we demonstrated that ß-elemene, elemene's primary component, effectively inhibits glycolysis in PCa cells by downregulating 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression. Furthermore, we found that ß-elemene accomplishes this downregulation by upregulating p53 and FZR1. Knockdown and overexpression experiments conclusively confirmed the pivotal role of PFKFB3 in mediating ß-elemene's anti-PCa activity. CONCLUSION: This finding presents compelling evidence that elemene exerts its anti-PCa effect by suppressing glycolysis through the downregulation of PFKFB3. This study not only improves our understanding of elemene in PCa treatment but also provides valuable insights for developing more effective and safer therapies for PCa.