Pyruvate Dehydrogenase Kinase Research Articles

Genetic Ablation of Pyruvate Dehydrogenase Kinase Isoform 4 Gene Enhances Recovery from Hyperoxic Lung Injury: Insights into Antioxidant and Inflammatory Mechanisms.

Pyruvate dehydrogenase kinase isoform 4 (PDK4) plays a pivotal role in the regulation of cellular proliferation and apoptosis. The objective of this study was to examine whether the genetic depletion of the PDK4 gene attenuates hyperoxia-induced lung injury in neonatal mice. Neonatal PDK4-/- mice and wild-type (WT) mice were exposed to oxygen concentrations of 21% (normoxia) and 95% (hyperoxia) for the first 4 days of life. Pulmonary histological assessments were performed, and the mRNA levels of lung PDK4, monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-6 were assessed. The levels of inflammatory cytokines in lung tissue were quantified. Following convalescence from neonatal hyperoxia, PDK4-/- mice exhibited improved lung alveolarization. Notably, PDK4-/- mice displayed significantly elevated MCP-1 protein levels in pulmonary tissues following 4 days of hyperoxic exposure, whereas WT mice showed increased IL-6 protein levels under similar conditions. Furthermore, neonatal PDK4-/- mice subjected to hyperoxia demonstrated markedly higher MCP-1 mRNA expression at 4 days of age compared to WT mice, while IL-6 mRNA expression remained unaffected in PDK4-/- mice. Newborn PDK4-/- mice exhibited notable recovery from hyperoxia-induced lung injury, suggesting the potential protective role of PDK4 depletion in mitigating lung damage.

Abstract 1784: Glucose metabolism and de novo palmitate synthesis under normoxia and hypoxia in breast cancer cells that preferentially metastasize to lung and liver

Abstract Cancer cells are exposed to variable oxygen and nutrient availability during the metastatic process which can influence metabolic adaptations of cancer cells to metastasize to distant sites. Thus, we investigated the metabolic adaptations of breast cancer cells that preferentially metastasize to lung (metM-WntLung cells; MLg) and liver (metM-WntLiver cells; MLr) in normoxia and hypoxia and at different glucose concentrations. In normoxia, 14C-glucose uptake is similar, but mRNA abundance of hexokinase, the initial rate-limiting step in glycolysis, was 22% higher in MLg cells. This is consistent with higher 13C6-glucose flux into glycolytic metabolites pyruvate (M+3) and lactate (M+3) in MLg compared to MLr. Also, de novo palmitate synthesis from 13C6-glucose was 7.6% higher in MLg and was accompanied by higher mRNA expression of ATP-citrate lyase (ACLY), which converts citrate to acetyl-CoA for fatty acid synthesis. These suggest greater glucose metabolism in MLg compared to MLr in normoxia, consistent with reduced viability of MLr by 39% in high glucose (25 mM) concentrations. Further, mRNA abundance of CPT1A, a rate-limiting step in fatty acid oxidation, is 23% higher in MLg. Moreover, the fatty acid oxidation inhibition (etomoxir) reduced viability of MLg by 26.7% compared to MLr, suggesting that fatty acid synthesis and oxidation are critical for MLg. Protein expression of HIF1α, a transcription factor that induces glycolysis during hypoxia, is 1.5-fold higher in MLr than MLg in normoxia, consistent with higher MLr mRNA abundance of pyruvate dehydrogenase kinase (PDK1), a target of HIF1α that inhibits pyruvate dehydrogenase (PDH) activity. Surprisingly, mRNA abundance of pyruvate carboxylase (PC), the enzyme that converts pyruvate to oxaloacetate, is also higher in MLr (39%) than MLg, thus supporting a potential alternative mechanism for glucose metabolites to enter the TCA cycle in normoxia in MLr. In hypoxia, viability of MLr, relative to MLg, increased by 11% at high glucose concentration. This was accompanied by higher hexokinase (33%) and PDK1 (83%) mRNA abundance suggesting MLr’s shift towards a glycolytic phenotype. However, de novo palmitate synthesis from 13C6-glucose is higher in MLg, suggesting that palmitate synthesis is maintained in MLg even in hypoxia. Altogether, results demonstrate that glucose utilization shifts in breast cancer cells that preferentially metastasize to lung vs liver, but de novo fatty acid synthesis is higher in MLg in different oxygen conditions. Thus, targeting breast cancer cells’ adaptation to glucose and de novo fatty acid synthesis may be a potential strategy to prevent breast cancer progression to distant organs. Citation Format: Marjorie Anne A. Layosa, Morgan Conrad, Stephen Hursting, Dorothy Teegarden. Glucose metabolism and de novo palmitate synthesis under normoxia and hypoxia in breast cancer cells that preferentially metastasize to lung and liver [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1784.

Alpha 2-adrenoceptor participates in anti-hyperalgesia by regulating metabolic demand.

The α2-adrenoceptor agonist dexmedetomidine is a commonly used drug for sedatives in clinics and has analgesic effects; however, its mechanism of analgesia in the spine remains unclear. In this study, we systematically used behavioural and transcriptomic sequencing, pharmacological intervention, electrophysiological recording and ultrasound imaging to explore the analgesic effects of the α2-adrenoceptor and its molecular mechanism. Firstly, we found that spinal nerve injury changed the spinal transcriptome expression, and the differential genes were mainly related to calcium signalling and tissue metabolic pathways. In addition, α2-adrenoceptor mRNA expression was significantly upregulated, and α2-adrenoceptor was significantly colocalised with markers, particularly neuronal markers. Intrathecal dexmedetomidine suppressed neuropathic pain and acute inflammatory pain in a dose-dependent manner. The transcriptome results demonstrated that the analgesic effect of dexmedetomidine may be related to the modulation of neuronal metabolism. Weighted gene correlation network analysis indicated that turquoise, brown, yellow and grey modules were the most correlated with dexmedetomidine-induced analgesic effects. Bioinformatics also annotated the involvement of metabolic processes and neural plasticity. A cardiovascular-mitochondrial interaction was found, and ultrasound imaging revealed that injection of dexmedetomidine significantly enhanced spinal cord perfusion in rats with neuropathic pain, which might be regulated by pyruvate dehydrogenase kinase 4 (pdk4), cholesterol 25-hydroxylase (ch25h) and GTP cyclohydrolase 1 (gch1). Increasing the perfusion doses of dexmedetomidine significantly suppressed the frequency and amplitude of spinal nerve ligation-induced miniature excitatory postsynaptic currents. Overall, dexmedetomidine exerts analgesic effects by restoring neuronal metabolic processes through agonism of the α2-adrenoceptor and subsequently inhibiting changes in synaptic plasticity.

Structure-based investigation of pyruvate dehydrogenase kinase-3 inhibitory potential of thymoquinone, targeting lung cancer therapy

Protein kinases are an attractive therapeutic target for cardiovascular, cancer and neurodegenerative diseases. Cancer cells demand energy generation through aerobic glycolysis, surpassing “oxidative phosphorylation” (OXPHOS) in mitochondria. The pyruvate dehydrogenase kinases (PDKs) have many regulatory roles in energy generation balance by controlling the pyruvate dehydrogenase complex. Overexpression of PDKs is associated with the overall survival of cancer. PDK3, an isoform of PDK is highly expressed in various cancer types, is targeted for inhibition in this study. PDK3 has been shown to binds strongly with a natural compound, thymoquinone (TQ), which is known to exhibit anti-cancer potential. Detailed interaction between the PDK3 and TQ was carried out using spectroscopic and docking methods. The overall changes in the protein's structures after TQ binding were estimated by UV–Vis spectroscopy, circular dichroism and fluorescence binding studies. The kinase activity assay was also carried out to see the kinase inhibitory potential of TQ. The enzyme inhibition assay suggested an excellent inhibitory potential of TQ towards PDK3 (IC50 = 5.49 μM). We observed that TQ forms a stable complex with PDK3 without altering its structure and can be a potent PDK3 inhibitor which may be implicated in cancer therapy after desired clinical validation.

Pan-cancer analysis reveals PDK family as potential indicators related to prognosis and immune infiltration

Pyruvate dehydrogenase kinases (PDKs) play a key role in glucose metabolism by exerting negative regulation over pyruvate dehyrogenase complex (PDC) activity through phosphorylation. Inhibition of PDKs holds the potential to enhance PDC activity, prompting cells to adopt a more aerobic metabolic profile. Consequently, PDKs emerge as promising targets for condition rooted in metabolic dysregulation, including malignance and diabetes. However, a comprehensive exploration of the distinct contribution of various PDK family members, particularly PDK3, across diverse tumor types remain incomplete. This study undertakes a systematic investigation of PDK family expression patterns, forging association with clinical parameters, using data from the TCGA and GTEx datasets. Survival analysis of PDKs is executed through both Kaplan–Meier analysis and COX regression analysis. Furthermore, the extent of immune infiltration is assessed by leveraging the CIBERSORT algorithm. Our study uncovers pronounced genetic heterogeneity among PDK family members, coupled with discernible clinical characteristic. Significantly, the study establishes the potential utility of PDK family genes as prognostic indicators and as predictors of therapeutic response. Additionally, our study sheds light on the immune infiltration profile of PDK family. The results showed the intimate involvement of these genes in immune-related metrics, including immune scoring, immune subtypes, tumor-infiltrating lymphocytes, and immune checkpoints expression. In sum, the findings of this study offer insightful strategies to guide the therapeutic direction, aiming at leveraging the impact of PDK family genes in cancer treatment.

LncRNA NORAD alleviates dysfunction of renal proximal tubular epithelial cells during the sepsis-associated acute kidney injury by modulating the miR-155-5p-PDK1 axis.

The sepsis-associated acute kidney injury (Sa-AKI) is closely related to high mortality rates worldwide. Injury to the renal proximal tubular epithelial cells (RPTECs), caused by pathological conditions, is a major cause of acute kidney injury (AKI). The lncRNA NORAD has been reported to be positively associated with kidney cancers. However, the biological roles and underlying mechanisms of NORAD in RPTECs during AKI are still unclear. In this study, we found that NORAD was significantly downregulated in RPTECs from AKI tissues. Overexpression of NORAD alleviated RPTECs injury induced by lipopolysaccharide (LPS). Additionally, glucose metabolism was significantly impaired during AKI, and LPS treatment inhibited glucose metabolism in RPTECs. We demonstrated that NORAD rescued the LPS-induced inhibition of glucose metabolism in RPTECs. Furthermore, miRNA-155-5p was significantly upregulated in RPTECs from AKI. Through bioinformatics analysis, RNA pull-down, RNA IP, and luciferase assays, we showed that NORAD directly associated with miR-155-5p to downregulate its expression. Moreover, overexpression of miR-155-5p inhibited glucose metabolism by directly targeting the 3'UTR of the glucose metabolism enzyme, pyruvate dehydrogenase kinase 1 (PDK1). Finally, rescue experiments validated that NORAD's protective effect on RPTECs injury was mediated through modulation of the miR-155-5p-PDK1-glucose metabolism pathway. In summary, these results reveal that lncRNA NORAD can alleviate RPTECs dysfunction by targeting the miR-155-5p-PDK1 axis, suggesting that NORAD has the potential to contribute to the development of therapeutic approaches against Sa-AKI.

Interleukin-6 classic and trans-signaling utilize glucose metabolism reprogramming to achieve anti- or pro-inflammatory effects

Interleukin (IL)-6 has anti- and pro-inflammatory functions, controlled by IL-6 classic and trans-signaling, respectively. Differences in the downstream signaling mechanism between IL-6 classic and trans-signaling have not been identified. Here, we report that IL-6 activates glycolysis to regulate the inflammatory response. IL-6 regulates glucose metabolism by forming a complex containing signal-transducing activators of transcription 3 (STAT3), hexokinase 2 (HK2), and voltage-dependent anion channel 1 (VDAC1). The IL-6 classic signaling directs glucose flux to oxidative phosphorylation (OxPhos), while IL-6 trans-signaling directs glucose flux to anaerobic glycolysis. Classic IL-6 signaling promotes STAT3 translocation into mitochondria to interact with pyruvate dehydrogenase kinase-1 (PDK1), leading to pyruvate dehydrogenase α (PDHA) dissociation from PDK1. As a result, PDHA is dephosphorylated, and STAT3 is phosphorylated at Ser727. By contrast, IL-6 trans-signaling promotes the interaction of sirtuin 2 (SIRT2) and lactate dehydrogenase A (LDHA), leading to the dissociation of STAT3 from SIRT2. As a result, LDHA is deacetylated, and STAT3 is acetylated and phosphorylated at Tyr705. IL-6 classic signaling promotes the differentiation of regulatory T cells via the PDK1/STAT3/PDHA axis, whereas IL-6 trans-signaling promotes the differentiation of Th17 cells via the SIRT2/STAT3/LDHA axis. Conclusion: IL-6 classic signaling generates anti-inflammatory functions by shifting energy metabolism to OxPhos, while IL-6 trans-signaling generates pro-inflammatory functions by shifting energy metabolism to anaerobic glycolysis.

Inhibition of Pyruvate Dehydrogenase Kinase 4 Protects Cardiomyocytes from lipopolysaccharide-Induced Mitochondrial Damage by Reducing Lactate Accumulation.

Mitochondrial dysfunction is considered one of the major pathogenic mechanisms of sepsis-induced cardiomyopathy (SIC). Pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of mitochondrial metabolism, is essential for maintaining mitochondrial function. However, its specific role in SIC remains unclear. To investigate this, we established an in vitro model of septic cardiomyopathy using lipopolysaccharide (LPS)-induced H9C2 cardiomyocytes. Our study revealed a significant increase in PDK4 expression in LPS-treated H9C2 cardiomyocytes. Inhibiting PDK4 with dichloroacetic acid (DCA) improved cell survival, reduced intracellular lipid accumulation and calcium overload, and restored mitochondrial structure and respiratory capacity while decreasing lactate accumulation. Similarly, Oxamate, a lactate dehydrogenase inhibitor, exhibited similar effects to DCA in LPS-treated H9C2 cardiomyocytes. To further validate whether PDK4 causes cardiomyocyte and mitochondrial damage in SIC by promoting lactate production, we upregulated PDK4 expression using PDK4-overexpressing lentivirus in H9C2 cardiomyocytes. This resulted in elevated lactate levels, impaired mitochondrial structure, and reduced mitochondrial respiratory capacity. However, inhibiting lactate production reversed the mitochondrial dysfunction caused by PDK4 upregulation. In conclusion, our study highlights the pathogenic role of PDK4 in LPS-induced cardiomyocyte and mitochondrial damage by promoting lactate production. Therefore, targeting PDK4 and its downstream product lactate may serve as promising therapeutic approaches for treating SIC.

Regulation and tumor-suppressive function of the miR-379/miR-656 (C14MC) cluster in cervical cancer.

Cervical cancer (CC) is a key contributor to cancer-related mortality in several countries. The identification of molecular markers and the underlying mechanism may help improve CC management. We studied the regulation and biological function of the chromosome 14 microRNA cluster (C14MC; miR-379/miR-656) in CC. Most C14MC members exhibited considerably lower expression in CC tissues and cell lines in The Cancer Genome Atlas (TCGA) cervical squamous cell carcinoma and endocervical adenocarcinoma patient cohorts. Bisulfite Sanger sequencing revealed hypermethylation of the C14MC promoter in CC tissues and cell lines. 5-aza-2 deoxy cytidine treatment reactivated expression of the C14MC members. We demonstrated that C14MC is a methylation-regulated miRNA cluster via artificial methylation and luciferase reporter assays. C14MC downregulation correlated with poor overall survival and may promote metastasis. C14MC activation via the lentiviral-based CRISPRa approach inhibited growth, proliferation, migration, and invasion; enhanced G2/M arrest; and induced senescence. Post-transcriptional regulatory network analysis of C14MC transcriptomic data revealed enrichment of key cancer-related pathways, such as metabolism, the cell cycle, and phosphatidylinositol 3-kinase (PI3K)-AKT signaling. Reduced cell proliferation, growth, migration, invasion, and senescence correlated with the downregulation of active AKT, MYC, and cyclin E1 (CCNE1) and the overexpression of p16, p21, and p27. We showed that C14MC miRNA activation increases reactive oxygen species (ROS) levels, intracellular Ca2+ levels, and lipid peroxidation rates, and inhibits epithelial-mesenchymal transition (EMT). C14MC targets pyruvate dehydrogenase kinase-3 (PDK3) according to the luciferase reporter assay. PDK3 is overexpressed in CC and is inversely correlated with C14MC. Both miR-494-mimic transfection and C14MC activation inhibited PDK3 expression. Reduced glucose uptake and lactate production, and upregulation of PDK3 upon C14MC activation suggest the potential role of these proteins in metabolic reprogramming. Finally, we showed that C14MC activation may inhibit EMT signaling. Thus, C14MC is a tumor-suppressive and methylation-regulated miRNA cluster in CC. Reactivation of C14MC can be useful in the management of CC.

Pyruvate dehydrogenase kinase 2 knockdown restores the ability of amyotrophic lateral sclerosis-linked SOD1G93A rat astrocytes to support motor neuron survival by increasing mitochondrial respiration.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration. Various studies using cellular and animal models of ALS indicate that there is a complex interplay between MN and neighboring non-neuronal cells, such as astrocytes, resulting in noncell autonomous neurodegeneration. Astrocytes in ALS exhibit a lower ability to support MN survival than nondisease-associated ones, which is strongly correlated with low-mitochondrial respiratory activity. Indeed, pharmacological inhibition of pyruvate dehydrogenase kinase (PDK) led to an increase in the mitochondrial oxidative phosphorylation pathway as the primary source of cell energy in SOD1G93A astrocytes and restored the survival of MN. Among the four PDK isoforms, PDK2 is ubiquitously expressed in astrocytes and presents low expression levels in neurons. Herein, we hypothesize whether selective knockdown of PDK2 in astrocytes may increase mitochondrial activity and, in turn, reduce SOD1G93A-associated toxicity. To assess this, cultured neonatal SOD1G93A rat astrocytes were incubated with specific PDK2 siRNA. This treatment resulted in a reduction of the enzyme expression with a concomitant decrease in the phosphorylation rate of the pyruvate dehydrogenase complex. In addition, PDK2-silenced SOD1G93A astrocytes exhibited restored mitochondrial bioenergetics parameters, adopting a more complex mitochondrial network. This treatment also decreased lipid droplet content in SOD1G93A astrocytes, suggesting a switch in energetic metabolism. Significantly, PDK2 knockdown increased the ability of SOD1G93A astrocytes to support MN survival, further supporting the major role of astrocyte mitochondrial respiratory activity in astrocyte-MN interactions. These results suggest that PDK2 silencing could be a cell-specific therapeutic tool to slow the progression of ALS.

Targeting the PDK/PDH axis to reverse metabolic abnormalities by structure-based virtual screening with in vitro and in vivo experiments

In humans and animals, the pyruvate dehydrogenase kinase (PDK) family proteins (PDKs 1–4) are excessively activated in metabolic disorders such as obesity, diabetes, and cancer, inhibiting the activity of pyruvate dehydrogenase (PDH) which plays a crucial role in energy and fatty acid metabolism and impairing its function. Intervention and regulation of PDH activity have become important research approaches for the treatment of various metabolic disorders.In this study, a small molecule (g25) targeting PDKs and activating PDH, was identified through multi-level computational screening methods. In vivo and in vitro experiments have shown that g25 activated the activity of PDH and reduced plasma lactate and triglyceride level. Besides, g25 significantly decreased hepatic fat deposition in a diet-induced obesity mouse model. Furthermore, g25 enhanced the tumor-inhibiting activity of cisplatin when used in combination. Molecular dynamics simulations and in vitro kinase assay also revealed the specificity of g25 towards PDK2.Overall, these findings emphasize the importance of targeting the PDK/PDH axis to regulate PDH enzyme activity in the treatment of metabolic disorders, providing directions for future related research. This study provides a possible lead compound for the PDK/PDH axis related diseases and offers insights into the regulatory mechanisms of this pathway in diseases.

PPP3CB Inhibits Cell Proliferation and the Warburg Effect in Bladder Cancer by Blocking PDHK1.

Cancer treatment has recently shifted towards metabolic approaches aimed at enhancing therapeutic efficacy. Somewhat surprisingly, a known regulator of energy metabolism in normal tissues, PPP3CB, is down-regulated in bladder cancer. This suggests that PPP3CB could exert an inhibitory effect on bladder cancer through its role in energy metabolism. To explore the above hypothesis, we employed non-targeted metabolism screening in bladder cancer cells with knockdown of PPP3CB. Glucose uptake and lactate production were carefully measured using specialized assay kits for glucose/lactic acid content. Western blot analysis was also used to evaluate the expression levels of pyruvate dehydrogenase kinase 1 (PDHK1) and p-PDHA1 in cells with PPP3CB knockdown. To substantiate the findings, co-immunoprecipitation (co-IP) experiments were performed to validate the interaction between PPP3CB and PDHK1. Various in vitro assays were also performed, including clone formation assay and Cell Counting Kit-8 (CCK8) viability assays. The in vivo anti-tumor potential of PPP3CB in bladder cancer was also studied using a nude mouse tumorigenesis model. Significant down-regulation of PPP3CB was observed in bladder tumors, and potent anti-tumor effects of PPP3CB were observed in vitro. Investigation of the underlying mechanism by which PPP3CB hampers glycolysis in bladder cancer cells revealed that it interacted with PDHK1 to inhibit its protein stabilization. PDHK1 thus appears to be a crucial mediator through which PPP3CB exerts its inhibitory effects on bladder cancer cells. In summary, PPP3CB exerts strong inhibitory influences on bladder cancer cell proliferation and glycolysis via its destabilization of PDHK1. These results highlight the potential of PPP3CB as a novel regulator of the Warburg effect. Interestingly, the downregulation of PPP3CB in bladder cancer cells increases the Warburg effect, thereby generating more lactic acid and reshaping the tumor microenvironment so as to promote tumor cell proliferation.

Reducing Oxidative Stress and Inflammation by Pyruvate Dehydrogenase Kinase 4 Inhibition Is Important in Prevention of Renal Ischemia-Reperfusion Injury in Diabetic Mice.

Reactive oxygen species (ROS) and inflammation are reported to have a fundamental role in the pathogenesis of ischemia-reperfusion (IR) injury, a leading cause of acute kidney injury. The present study investigated the role of pyruvate dehydrogenase kinase 4 (PDK4) in ROS production and inflammation following IR injury. We used a streptozotocin-induced diabetic C57BL6/J mouse model, which was subjected to IR by clamping both renal pedicles. Cellular apoptosis and inflammatory markers were evaluated in NRK-52E cells and mouse primary tubular cells after hypoxia and reoxygenation using a hypoxia work station. Following IR injury in diabetic mice, the expression of PDK4, rather than the other PDK isoforms, was induced with a marked increase in pyruvate dehydrogenase E1α (PDHE1α) phosphorylation. This was accompanied by a pronounced ROS activation, as well as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1) production. Notably, sodium dichloroacetate (DCA) attenuated renal IR injury-induced apoptosis which can be attributed to reducing PDK4 expression and PDHE1α phosphorylation levels. DCA or shPdk4 treatment reduced oxidative stress and decreased TNF-α, IL-6, IL-1β, and MCP-1 production after IR or hypoxia-reoxygenation injury. PDK4 inhibition alleviated renal injury with decreased ROS production and inflammation, supporting a critical role for PDK4 in IR mediated damage. This result indicates another potential target for reno-protection during IR injury; accordingly, the role of PDK4 inhibition needs to be comprehensively elucidated in terms of mitochondrial function during renal IR injury.

Design, synthesis and in vitro antitumor activity of 17β-estradiol-amino acid derivatives

A total of 28 derivatives were designed and synthesized, including 18 estradiol-amino acid (17β-OH) derivatives (I-2∼I-19) and 10 estradiol-amino acid (16-C) derivatives (II-2∼II-11). Amino acids used in synthesis include L-phenylalanine, L-serine, L-leucine, glycine, L-proline and L-glutamine. The results showed that I-8∼I-12 of 17β-OH conjugated amino acids in 17β-estradiol had good in vitro antitumor activity, indicating that the introduction of amino acids could improve the anti-proliferative effect. Among them, 3-benzyloxy-Estra-1,3,5 (10)-triene-17β-ol pyrrolidine-2-carboxylate hydrochloride (I-12) showed the best inhibitory activity against breast cancer (MDA-MB-231) cells (IC50 = 4.89 µM). Further studies on apoptosis revealed that I-12 could regulate the expression of apoptosis-related proteins in MDA-MB-231 cells. Network pharmacology and molecular docking analysis revealed that I-12 may act on targets such as mechanistic target of rapamycin kinase (mTOR), estrogen receptor 1 (ESR1), pyruvate dehydrogenase kinase (PDK2) targets and their related pathways.

Hypoxic Signaling Pathways in Carotid Body Tumors

Carotid body tumors (CBTs) are rare tumors with a 1–2 incidence per 100,000 individuals. CBTs may initially present without apparent symptoms, and symptoms begin to arise since tumors grow bigger to compress surrounding tissue, such as recurrent laryngeal nerve and esophagus. Also, the etiology of CBTs remains unclear since it is more likely to occur in those who live in high-altitude areas or suffer from chronic hypoxic diseases such as COPD. SDH mutations and familial inheritance have been reported to be related to CBTs. SDH complexes play crucial roles in aerobic respiration, and SDH mutations in CBTs have been reported to be associated with hypoxia. Hypoxic signaling pathways, specifically hypoxic markers, have attracted more research attention in tumor exploration. However, the existing literature on these signaling and markers lacks a systematic review. Also, therapeutic approaches in CBTs based on hypoxic signaling are rarely used in clinics. In this review, we concluded the role of hypoxic signaling and markers and their potential implications in the initiation and progression of CBTs. Our findings underscore the involvement of the SDH family, the HIF family, VEGFs, and inflammatory cytokines (ICs) in tumorigenesis and treatment. Of particular interest is the role played by SDHx, which has recently been linked to oxygen sensing through mutations leading to hereditary CBTs. Among the SDH family, SDHB and SDHD exhibit remarkable characteristics associated with metastasis and multiple tumors. Besides SDH mutations in CBTs, the HIF family also plays crucial roles in CBTs via hypoxic signaling pathways. The HIF family regulates angiogenesis during mammalian development and tumor growth by gene expression in CBTs. HIF1α could induce the transcription of pyruvate dehydrogenase kinase 1 (PDK1) to inhibit pyruvate dehydrogenase kinase (PDH) by inhibiting the TCA cycle. Then, carotid body cells begin to hyperplasia and hypertrophy. At the same time, EPAS1 mutation, an activating mutation, could decrease the degradation of HIF2α and result in Pacak–Zhuang syndrome, which could result in paraganglioma. HIFs can also activate VEGF expression, and VEGFs act on Flk-1 to control the hyperplasia of type I cells and promote neovascularization. ICs also play a pivotal signaling role within the CB, as their expression is induced under hypoxic conditions to stimulate CB hyperplasia, ultimately leading to CBTs detecting hypoxic areas in tumors, and improving the hypoxic condition could enhance photon radiotherapy efficacy. Moreover, this review offers valuable insights for future research directions on understanding the relationship between hypoxic signaling pathways and CBTs.

Disruption of mitochondrial pyruvate oxidation in dorsal root ganglia drives persistent nociceptive sensitization and causes pervasive transcriptomic alterations.

Metabolism is inextricably linked to every aspect of cellular function. In addition to energy production and biosynthesis, metabolism plays a crucial role in regulating signal transduction and gene expression. Altered metabolic states have been shown to maintain aberrant signaling and transcription, contributing to diseases like cancer, cardiovascular disease, and neurodegeneration. Metabolic gene polymorphisms and defects are also associated with chronic pain conditions, as are increased levels of nerve growth factor (NGF). However, the mechanisms by which NGF may modulate sensory neuron metabolism remain unclear. This study demonstrated that intraplantar NGF injection reprograms sensory neuron metabolism. Nerve growth factor suppressed mitochondrial pyruvate oxidation and enhanced lactate extrusion, requiring 24 hours to increase lactate dehydrogenase A and pyruvate dehydrogenase kinase 1 (PDHK1) expression. Inhibiting these metabolic enzymes reversed NGF-mediated effects. Remarkably, directly disrupting mitochondrial pyruvate oxidation induced severe, persistent allodynia, implicating this metabolic dysfunction in chronic pain. Nanopore long-read sequencing of poly(A) mRNA uncovered extensive transcriptomic changes upon metabolic disruption, including altered gene expression, splicing, and poly(A) tail lengths. By linking metabolic disturbance of dorsal root ganglia to transcriptome reprogramming, this study enhances our understanding of the mechanisms underlying persistent nociceptive sensitization. These findings imply that impaired mitochondrial pyruvate oxidation may drive chronic pain, possibly by impacting transcriptomic regulation. Exploring these metabolite-driven mechanisms further might reveal novel therapeutic targets for intractable pain.

Investigating the role of thymol as a promising inhibitor of pyruvate dehydrogenase kinase 3 for targeted cancer therapy

Protein kinases have emerged as major contributors to various diseases. They are currently exploited as a potential target in drug discovery because they play crucial roles in cell signaling, growth, and regulation. Their dysregulation is associated with inflammatory disorders, cancer, and neurodegenerative diseases. Pyruvate dehydrogenase kinase 3 (PDK3) has become an attractive drug target in cancer therapeutics. In the present study, we investigated the effective role of thymol in PDK3 inhibition due to the high affinity predicted through molecular docking studies. Hence, to better understand this inhibition mechanism, we carried out a 100 ns molecular dynamics (MD) simulation to analyse the dynamics and stability of the PDK3-thymol complex. The PDK3-thymol complex was stable and energetically favourable, with many intramolecular hydrogen bond interactions in the PDK3-thymol complex. Enzyme inhibition assay showed significant inhibition of PDK3 by thymol, revealing potential inhibitory action of thymol towards PDK3 (IC50 = 2.66 μM). In summary, we established thymol as one of the potential inhibitors of PDK3, proposing promising therapeutic implications for severe diseases associated with PDK3 dysregulation. This study further advances our understanding of thymol's therapeutic capabilities and potential role in cancer treatment.

Natural Product-Based Glycolysis Inhibitors as a Therapeutic Strategy for Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor-Resistant Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide. Targeted therapy against the epidermal growth factor receptor (EGFR) is a promising treatment approach for NSCLC. However, resistance to EGFR tyrosine kinase inhibitors (TKIs) remains a major challenge in its clinical management. EGFR mutation elevates the expression of hypoxia-inducible factor-1 alpha to upregulate the production of glycolytic enzymes, increasing glycolysis and tumor resistance. The inhibition of glycolysis can be a potential strategy for overcoming EGFR-TKI resistance and enhancing the effectiveness of EGFR-TKIs. In this review, we specifically explored the effectiveness of pyruvate dehydrogenase kinase inhibitors and lactate dehydrogenase A inhibitors in combating EGFR-TKI resistance. The aim was to summarize the effects of these natural products in preclinical NSCLC models to provide a comprehensive understanding of the potential therapeutic effects. The study findings suggest that natural products can be promising inhibitors of glycolytic enzymes for the treatment of EGFR-TKI-resistant NSCLC. Further investigations through preclinical and clinical studies are required to validate the efficacy of natural product-based glycolytic inhibitors as innovative therapeutic modalities for NSCLC.

Circadian modulation of glucose utilization via CRY1-mediated repression of Pdk1 expression

Life adapts to daily environmental changes through circadian rhythms, exhibiting spontaneous oscillations of biological processes. These daily functional oscillations must match the metabolic requirements responding to the time of the day. We focus on the molecular mechanism of how the circadian clock regulates glucose, the primary resource for energy production and other biosynthetic pathways. The complex regulation of the circadian rhythm includes many proteins that control this process at the transcriptional and translational levels and by protein-protein interactions. We have investigated the action of one of these proteins, Cryptochrome (CRY), whose elevated mRNA and protein levels repress the function of an activator in the transcription-translation feedback loop, and this activator causes elevated Cry1 mRNA. We used a genome-edited cell line model to investigate downstream genes affected explicitly by the repressor CRY. We found that CRY can repress glycolytic genes, particularly that of the gatekeeper, pyruvate dehydrogenase kinase 1 (Pdk1), decreasing lactate accumulation and glucose utilization. CRY1-mediated decrease of Pdk1 expression can also be observed in a breast cancer cell line MDA-MB-231, whose glycolysis is associated with Pdk1 expression. We also found that exogenous expression of CRY1 in the MDA-MB-231 decreases glucose usage and growth rate. Furthermore, reduced CRY1 levels and the increased phosphorylation of PDK1 substrate were observed when cells were grown in suspension compared to cells grown in adhesion. Our data supports a model that the transcription-translation feedback loop can regulate the glucose metabolic pathway through Pdk1 gene expression according to the time of the day.

Melatonin: a modulator in metabolic rewiring in T-cell malignancies.

Melatonin, (N-acetyl-5-methoxytryptamine) an indoleamine exerts multifaced effects and regulates numerous cellular pathways and molecular targets associated with circadian rhythm, immune modulation, and seasonal reproduction including metabolic rewiring during T cell malignancy. T-cell malignancies encompass a group of hematological cancers characterized by the uncontrolled growth and proliferation of malignant T-cells. These cancer cells exhibit a distinct metabolic adaptation, a hallmark of cancer in general, as they rewire their metabolic pathways to meet the heightened energy requirements and biosynthesis necessary for malignancies is the Warburg effect, characterized by a shift towards glycolysis, even when oxygen is available. In addition, T-cell malignancies cause metabolic shift by inhibiting the enzyme pyruvate Dehydrogenase Kinase (PDK) which in turn results in increased acetyl CoA enzyme production and cellular glycolytic activity. Further, melatonin plays a modulatory role in the expression of essential transporters (Glut1, Glut2) responsible for nutrient uptake and metabolic rewiring, such as glucose and amino acid transporters in T-cells. This modulation significantly impacts the metabolic profile of T-cells, consequently affecting their differentiation. Furthermore, melatonin has been found to regulate the expression of critical signaling molecules involved in T-cell activations, such as CD38, and CD69. These molecules are integral to T-cell adhesion, signaling, and activation. This review aims to provide insights into the mechanism of melatonin's anticancer properties concerning metabolic rewiring during T-cell malignancy. The present review encompasses the involvement of oncogenic factors, the tumor microenvironment and metabolic alteration, hallmarks, metabolic reprogramming, and the anti-oncogenic/oncostatic impact of melatonin on various cancer cells.