Triptolide restrains the growth, invasion, stemness, and glycolysis of non-small cell lung cancer cells by PFKFB2-mediated PI3K/AKT pathway.

Triptolide (TP) has been found to have anti-tumor effects. However, more potential molecular mechanisms of TP in the progression of non-small cell lung cancer (NSCLC) deserve further investigation. Cell proliferation, apoptosis, invasion, and stemness were detected by cell counting kit 8 assay, EdU assay, flow cytometry, transwell assay, and sphere formation assay. Cell glycolysis was evaluated by corresponding assay kits. 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2) expression was measured by western blot (WB), qRT-PCR and immunohistochemical staining. PI3K/AKT pathway-related markers were determined by WB. Besides, xenograft tumor model was conducted to evaluate the anti-tumor effect of TP in NSCLC. Our results revealed that TP treatment suppressed NSCLC cell proliferation, invasion, stemness, glycolysis, and enhanced apoptosis. PFKFB2 was upregulated in NSCLC tissues and cells, and its expression was decreased by TP. PFKFB2 knockdown restrained NSCLC cell functions, and its overexpression also eliminated TP-mediated NSCLC cell functions inhibition. TP decreased PFKFB2 expression to inactivate PI3K/AKT pathway. Moreover, PI3K/AKT pathway inhibitor LY294002 also could reverse the promoting effect of PFKFB2 on NSCLC cell functions. In addition, TP suppressed NSCLC tumorigenesis by inhibiting PFKFB2/PI3K/AKT pathway. In conclusion, TP exerted anti-tumor role in NSCLC, which was achieved by reducing PFKFB2 expression to inactivate PI3K/AKT pathway.

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.

TRAF7 inhibits glycolysis to potentiate growth inhibition and apoptosis of myeloid leukemia cells via regulating the KLF2-PFKFB3 axis.

Tumor necrosis factor receptor-related factor 7 (TRAF7) can regulate cell differentiation and apoptosis, but its specific functional mechanism in the pathological process of acute myeloid leukemia (AML) closely related to differentiation and apoptosis disorders is largely unclear. In this study, TRAF7 was found to be lowly expressed in AML patients and a variety of myeloid leukemia cells. TRAF7 was overexpressed in AML Molm-13 and chronic myeloid leukemia (CML) K562 cells by transfection with pcDNA3.1-TRAF7. CCK-8 assay and flow cytometry analysis showed that TRAF7 overexpression induced growth inhibition and apoptosis in K562 and Molm-13 cells. Measurements of glucose and lactate suggested that TRAF7 overexpression impaired glycolysis of K562 and Molm-13 cells. Cell cycle analysis indicated that most of K562 and Molm-13 cells were captured in G0/G1 phase by TRAF7 overexpression. PCR and western blot assay revealed that TRAF7 increased Kruppel-like factor 2 (KLF2) expression but decreased 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression in AML cells. KLF2 knockdown can counteract TRAF7-triggered PFKFB3 inhibition, and abolish TRAF7-mediated glycolysis inhibition and cell cycle arrest. KLF2 knockdown or PFKFB3 overexpression both can partially neutralize TRAF7-induced growth inhibition and apoptosis of K562 and Molm-13 cells. Moreover, Lv-TRAF7 decreased human CD45+ cells in mouse peripheral blood in the xenograft mice established by NOD/SCID mice. Taken together, TRAF7 exerts anti-leukemia effects by impairing glycolysis and cell cycle progression of myeloid leukemia cells via modulating the KLF2-PFKFB3 axis.

Specific PFKFB3 Inhibitor Memorably Ameliorates Intervertebral Disc Degeneration via Inhibiting NF-κB and MAPK Signaling Pathway and Reprogramming of Energy Metabolism of Nucleus Pulposus Cells.

Intervertebral disc (IVD) degeneration (IVDD) is a characteristic of the dominating pathological processes of nucleus pulposus (NP) cell senescence, abnormal synthesis and irregular distribution of extracellular matrix (ECM), and tumor necrosis factor-α (TNF-α) induced inflammation. Nowadays, IVD acid environment variation which accelerates the pathological processes mentioned above arouses researchers' attention. KAN0438757 (KAN) is an effective inhibitor of selective metabolic kinase phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) that has both energy metabolism reprogramming and anti-inflammatory effects. Therefore, a potential therapeutic benefit of KAN lies in its ability to inhibit the development of IVDD. This study examined in vitro KAN toxicity in NP primary cells (NPPs). Moreover, KAN influenced tumor necrosis factor-α (TNF-α) induced ECM anabolism and catabolism; the inflammatory signaling pathway activation and the energy metabolism phenotype were also examined in NPPs. Furthermore, KAN's therapeutic effect was investigated in vivo using the rat tail disc puncture model. Phenotypically speaking, the KAN treatment partially rescued the ECM degradation and glycolysis energy metabolism phenotypes of NPPs induced by TNF-α. In terms of mechanism, KAN inhibited the activation of MAPK and NF-κB inflammatory signaling pathways induced by TNF-α and reprogramed the energy metabolism. For the therapeutic aspect, the rat tail disc puncture model demonstrated that KAN has a significant ameliorated effect on the progression of IVDD. To sum up, our research successfully authenticated the potential therapeutic effect of KAN on IVDD and declaimed its mechanisms of both novel energy metabolism reprogramming and conventional anti-inflammation effect.

AAV-mediated expression of PFKFB3 in myofibers, but not endothelial cells, improves ischemic muscle function in mice with critical limb ischemia.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) is a powerful driver of angiogenesis through its modulation of glycolytic metabolism within endothelial cells. Recent work has demonstrated that PFKFB3 modulates the response to muscle ischemia, however the cell specificity of these effects is not fully understood. In this study, we tested the impact of viral mediated expression of PFKFB3, driven by gene promoters specific for myofibers or endothelial cells, on ischemic hindlimb revascularization and muscle function. We hypothesized that both endothelium- and muscle-specific expression of PFKFB3 would attenuate limb pathology following femoral artery ligation. Male and female BALB/cJ mice were injected with adeno-associated virus encoding the either a green fluorescent protein (GFP) or PFKFB3 driven by either the human skeletal actin (ACTA1) or cadherin-5 (Cdh5) promoters. Four weeks after AAV treatment, mice were subjected to unilateral femoral artery ligation and limb perfusion and muscle function were assessed. Both endothelium- and muscle-specific PFKFB3 expression resulted in significantly more perfused capillaries within the ischemic limb muscle, but neither changed myofiber size/area. Muscle-specific, but not endothelium-specific, PFKFB3 expression significantly improved maximal force production in ischemic muscle (P = 0.0005). Notably, there was a significant effect of sex on maximal force levels in both cohorts of mice (P = 0.0075 and P = 0.0481), indicating that female mice had higher ischemic muscle strength compared with male mice, regardless of treatment group. Taken together, these data demonstrate that although both muscle- and endothelium-specific expression of PFKFB3 enhanced ischemic revascularization, only muscle-specific PFKFB3 expression improved muscle function.NEW & NOTEWORTHY Critical limb ischemia (CLI) carries a significant risk for limb amputation, and treatment options remain limited. We tested the impact of expression of PFKFB3 in myofibers or endothelial cells on limb pathology in mice with CLI. Although both muscle and endothelium-specific PFKFB3 expression increased perfused capillary density, only muscle-specific PFKFB3 expression improve contractile function. Regardless of treatment, female mice demonstrated better recovery from limb ischemic compared with male mice.

Inhibition of PFKFB3 in HER2-positive gastric cancer improves sensitivity to trastuzumab by inducing tumour vessel normalisation.

BACKGROUND: Multiple mechanisms have been proposed that lead to reduced effectiveness of trastuzumab in HER2-positive gastric cancer (GC), yet resistance to trastuzumab remains a challenge in clinics. METHODS: We established trastuzumab-resistant cells and patient-derived xenografts models to measure metabolic levels and vascular density and shape. The HER2-positive GC patient samples were used to determine clinical significance. We also measured protein expression and phosphorylation modifications to determine those alterations related to resistance. In vivo studies combining inhibitor of PFKFB3 with trastuzumab corroborated the in vitro findings. RESULTS: The 6-phosphofructo-2-kinase (PFKFB3)-mediated trastuzumab resistance pathways in HER2-positive GC by activating the glycolytic pathway. We also found vessels are chaotic and destabilised in the tumour during the trastuzumab resistance process. Inhibition of PFKFB3 significantly diminished tumour proliferation and promoted vessel normalisation in the patient-derived xenograft model. Mechanistically, PFKFB3 promoted the secretion of CXCL8 into the tumour microenvironment, and phosphorylated Ser1151 of ERBB2, enhancing the transcription of CXCL8 by activating the PI3K/AKT/NFκB p65 pathway. CONCLUSIONS: Our current findings discover that PFKFB3 inhibitors might be effective tools to overcome adjuvant therapy resistance in HER2-positive GC and reshaping the microenvironment by normalising tumour vessels is a novel strategy to overcome trastuzumab resistance.

Studying the Anti-Tumor Effects of siRNA Gene Silencing of Some Metabolic Genes in Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Earlier diagnosis and advances in treatment strategies have increased the average survival of cancer patients over the last decades. Despite the increased number of new anti- neoplastic agents, there has been no adequate therapy for intricate malignancies, such as pancreatic cancer. Cancer metabolism is the main building block standing behind cancer promotion and progression, even in the presence of a harsh environment. Targeting metabolic pathways, such as glycolysis and pentose phosphate pathway, are regarded as a promising new strategy for cancer treatment. OBJECTIVE: The current study aimed to investigate the effects of knocking-down pancreatic cancer glycolytic and pentose phosphate pathway's regulators (HIF-1α, ARNT, PFKFB4, and RBKS) on cell's viability and resistance to Gemcitabine and Doxorubicin, using small interference RNA. METHODOLOGY: The human pancreatic ductal adenocarcinoma cell line, Panc-1, was used to study the anti-proliferative activity of targeting HIF-1α, ARNT, PFKFB4, and RBKS mRNAs by transfection with small interference RNAs, alone and in combination. The transfected cells were also treated with Doxorubicin and Gemcitabine to study the relationship between the concerned genes and the resistance of Panc-1 cells to these drugs. The effect on cell proliferation was determined using a colorimetric assay and Inhibitory Concentration ((IC50) calculation. A cross-talk study was done to investigate the silencing effect of one of the above genes on the expression of others using Real Time-Polymerase Chain Reaction. RESULTS: In vitro transfection with small interference-RNAs, siHIF-1α, siPFKFB4, and siARNT decreased tumor cell proliferation with a maximum effect shown by siPFKFB4; but there was no anti- proliferative effect with RBKS silencing. Suppression of transcription of HIF-1α, ARNT, PFKFB4, and RBKS sensitize pancreatic cancer cells, Panc-1, to Doxorubicin and Gemcitabine. CONCLUSION: This study demonstrated the major tumor-promoting and progressive effects of PFKFB4, while HIF-1α and ARNT had modulator effects in pancreatic cancer cells (Panc-1). RBKS had a chemo-resistant role, justifying its enhanced expression in Panc-1 cells, but not a proliferative one. Silencing of all genes of interest decreased Doxorubicin and Gemcitabine's resistance and improved their antitumor effect Din the pancreatic cancer cell line, Panc-1.

Discover potential inhibitors for PFKFB3 using 3D-QSAR, virtual screening, molecular docking and molecular dynamics simulation.

The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) is a master regulator of glycolysis in cancer cells by synthesizing fructose-2,6-bisphosphate (F-2,6-BP), a potent allosteric activator of phosphofructokinase-1 (PFK-1), which is a rate-limiting enzyme of glycolysis. PFKFB3 is an attractive target for cancer treatment. It is valuable to discover promising inhibitors by using 3D-QSAR pharmacophore modeling, virtual screening, molecular docking and molecular dynamics simulation. Twenty molecules with known activity were used to build 3D-QSAR pharmacophore models. The best pharmacophore model was ADHR called Hypo1, which had the highest correlation value of 0.98 and the lowest RMSD of 0.82. Then, the Hypo1 was validated by cost value method, test set method and decoy set validation method. Next, the Hypo1 combined with Lipinski's rule of five and ADMET properties were employed to screen databases including Asinex and Specs, total of 1,048,159 molecules. The hits retrieved from screening were docked into protein by different procedures including HTVS, SP and XP. Finally, nine molecules were picked out as potential PFKFB3 inhibitors. The stability of PFKFB3-lead complexes was verified by 40 ns molecular dynamics simulation. The binding free energy and the energy contribution of per residue to the binding energy were calculated by MM-PBSA based on molecular dynamics simulation.

Increasing fructose 2,6-bisphosphate overcomes hepatic insulin resistance of type 2 diabetes.

Hepatic glucose production is increased as a metabolic consequence of insulin resistance in type 2 diabetes. Because fructose 2,6-bisphosphate is an important regulator of hepatic glucose production, we used adenovirus-mediated enzyme overexpression to increase hepatic fructose 2,6-bisphosphate to determine if the hyperglycemia in KK mice, polygenic models of type 2 diabetes, could be ameliorated by reduction of hepatic glucose production. Seven days after treatment with virus encoding a mutant 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase designed to increase fructose 2,6-bisphosphate levels, plasma glucose, lipids, and insulin were significantly reduced in KK/H1J and KK.Cg-A(y)/J mice. Moreover, high fructose 2,6-bisphosphate levels downregulated glucose-6-phosphatase and upregulated glucokinase gene expression, thereby reversing the insulin-resistant pattern of hepatic gene expression of these two key glucose-metabolic enzymes. The increased hepatic fructose 2,6-bisphosphate also reduced adiposity in both KK mice. These results clearly indicate that increasing hepatic fructose 2,6-bisphosphate overcomes the impairment of insulin in suppressing hepatic glucose production, and it provides a potential therapy for type 2 diabetes.