High expression of phosphoglycerate dehydrogenase predicts poor outcome in patients with high-grade serous ovarian cancer.

INTRODUCTION: High-grade serous ovarian cancer (HGSOC) is characterized by high mortality and prevalent recurrences. This study investigates the prognostic value of phosphoglycerate dehydrogenase (PHGDH) in HGSOC which has been linked to metabolic reprogramming and recurrences in other cancers. METHODS: Data from 306 patients with advanced-stage HGSOC treated between 2008 and 2015 were analyzed. PHGDH expression levels were determined using immunohistochemistry and categorized as "low" or "high." RESULTS: PHGDH-high was associated with higher FIGO stage and increased use of neoadjuvant chemotherapy. Patients with PHGDH-high tumors had significantly worse survival than PHDH-low, even after adjusting for confounding factors.

Is allostery a fuzzy concept?

Allostery is an important property of biological macromolecules which regulates diverse biological functions such as catalysis, signal transduction, transport, and molecular recognition. However, the concept was expressed using two different definitions by J. Monod and, over time, more have been added by different authors, making it fuzzy. Here, we reviewed the different meanings of allostery in the current literature and found that it has been used to indicate that the function of a protein is regulated by heterotropic ligands, and/or that the binding of ligands and substrates presents homotropic positive or negative cooperativity, whatever the hypothesized or demonstrated reaction mechanism might be. Thus, proteins defined to be allosteric include not only those that obey the two-state concerted model, but also those that obey different reaction mechanisms such as ligand-induced fit, possibly coupled to sequential structure changes, and ligand-linked dissociation-association. Since each reaction mechanism requires its own mathematical description and is defined by it, there are many possible 'allosteries'. This lack of clarity is made even fuzzier by the fact that the reaction mechanism is often assigned imprecisely and/or implicitly in the absence of the necessary experimental evidence. In this review, we examine a list of proteins that have been defined to be allosteric and attempt to assign a reaction mechanism to as many as possible.

Yap/Taz activity is associated with increased expression of phosphoglycerate dehydrogenase that supports myoblast proliferation.

In skeletal muscle, the Hippo effector Yap promotes satellite cell, myoblast, and rhabdomyoblast proliferation but prevents myogenic differentiation into multinucleated muscle fibres. We previously noted that Yap drives expression of the first enzyme of the serine biosynthesis pathway, phosphoglycerate dehydrogenase (Phgdh). Here, we examined the regulation and function of Phgdh in satellite cells and myoblasts and found that Phgdh protein increased during satellite cell activation. Analysis of published data reveal that Phgdh mRNA in mouse tibialis anterior muscle was highly expressed at day 3 of regeneration after cardiotoxin injection, when markers of proliferation are also robustly expressed and in the first week of synergist-ablated muscle. Finally, siRNA-mediated knockdown of PHGDH significantly reduced myoblast numbers and the proliferation rate. Collectively, our data suggest that Phgdh is a proliferation-enhancing metabolic enzyme that is induced when quiescent satellite cells become activated.

Homoharringtonine as a PHGDH inhibitor: Unraveling metabolic dependencies and developing a potent therapeutic strategy for high-risk neuroblastoma.

Neuroblastoma, a childhood cancer affecting the sympathetic nervous system, continues to challenge the development of potent treatments due to the limited availability of druggable targets for this aggressive illness. Recent investigations have uncovered that phosphoglycerate dehydrogenase (PHGDH), an essential enzyme for de novo serine synthesis, serves as a non-oncogene dependency in high-risk neuroblastoma. In this study, we show that homoharringtonine (HHT) acts as a PHGDH inhibitor, inducing intricate alterations in cellular metabolism, and thus providing an efficient treatment for neuroblastoma. We have experimentally verified the reliance of neuroblastoma on PHGDH and employed molecular docking, thermodynamic evaluations, and X-ray crystallography techniques to determine the bond interactions between HHT and PHGDH. Administering HHT to treat neuroblastoma resulted in effective cell elimination in vitro and tumor reduction in vivo. Metabolite and functional assessments additionally disclosed that HHT treatment suppressed de novo serine synthesis, initiating intricate metabolic reconfiguration and oxidative stress in neuroblastoma. Collectively, these discoveries highlight the potential of targeting PHGDH using HHT as a potent approach for managing high-risk neuroblastoma.

MicroRNA regulation of the serine synthesis pathway in endocrine-resistant breast cancer cells.

Despite the successful combination of therapies improving survival of estrogen receptor α (ER+) breast cancer patients with metastatic disease, mechanisms for acquired endocrine resistance remain to be fully elucidated. The RNA binding protein HNRNPA2B1 (A2B1), a reader of N(6)-methyladenosine (m6A) in transcribed RNA, is upregulated in endocrine-resistant, ER+ LCC9 and LY2 cells compared to parental MCF-7 endocrine-sensitive luminal A breast cancer cells. The miRNA-seq transcriptome of MCF-7 cells overexpressing A2B1 identified the serine metabolic processes pathway. Increased expression of two key enzymes in the serine synthesis pathway (SSP), phosphoserine aminotransferase 1 (PSAT1) and phosphoglycerate dehydrogenase (PHGDH), correlates with poor outcomes in ER+ breast patients who received tamoxifen (TAM). We reported that PSAT1 and PHGDH were higher in LCC9 and LY2 cells compared to MCF-7 cells and their knockdown enhanced TAM sensitivity in these-resistant cells. Here we demonstrate that stable, modest overexpression of A2B1 in MCF-7 cells increased PSAT1 and PHGDH and endocrine resistance. We identified four miRNAs downregulated in MCF-7-A2B1 cells that directly target the PSAT1 3'UTR (miR-145-5p and miR-424-5p), and the PHGDH 3'UTR (miR-34b-5p and miR-876-5p) in dual luciferase assays. Lower expression of miR-145-5p and miR-424-5p in LCC9 and ZR-75-1-4-OHT cells correlated with increased PSAT1 and lower expression of miR-34b-5p and miR-876-5p in LCC9 and ZR-75-1-4-OHT cells correlated with increased PHGDH. Transient transfection of these miRNAs restored endocrine-therapy sensitivity in LCC9 and ZR-75-1-4-OHT cells. Overall, our data suggest a role for decreased A2B1-regulated miRNAs in endocrine resistance and upregulation of the SSP to promote tumor progression in ER+ breast cancer.

Perturbation of serine enantiomers homeostasis in the striatum of MPTP-lesioned monkeys and mice reflects the extent of dopaminergic midbrain degeneration.

Loss of dopaminergic midbrain neurons perturbs l-serine and d-serine homeostasis in the post-mortem caudate putamen (CPu) of Parkinson's disease (PD) patients. However, it is unclear whether the severity of dopaminergic nigrostriatal degeneration plays a role in deregulating serine enantiomers' metabolism. Here, through high-performance liquid chromatography (HPLC), we measured the levels of these amino acids in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys and MPTP-plus-probenecid (MPTPp)-treated mice to determine whether and how dopaminergic midbrain degeneration affects the levels of serine enantiomers in various basal ganglia subregions. In addition, in the same brain regions, we measured the levels of key neuroactive amino acids modulating glutamatergic neurotransmission, including l-glutamate, glycine, l-aspartate, d-aspartate, and their precursors l-glutamine, l-asparagine. In monkeys, MPTP treatment produced severe denervation of nigrostriatal dopaminergic fibers (⁓75%) and increased the levels of serine enantiomers in the rostral putamen (rPut), but not in the subthalamic nucleus, and the lateral and medial portion of the globus pallidus. Moreover, this neurotoxin significantly reduced the protein expression of the astrocytic serine transporter ASCT1 and the glycolytic enzyme GAPDH in the rPut of monkeys. Conversely, concentrations of d-serine and l-serine, as well as ASCT1 and GAPDH expression were unaffected in the striatum of MPTPp-treated mice, which showed only mild dopaminergic degeneration (⁓30%). These findings unveil a link between the severity of dopaminergic nigrostriatal degeneration and striatal serine enantiomers concentration, ASCT1 and GAPDH expression. We hypothesize that the up-regulation of d-serine and l-serine levels occurs as a secondary response within a homeostatic loop to support the metabolic and neurotransmission demands imposed by the degeneration of dopaminergic neurons.

PHGDH preserves one-carbon cycle to confer metabolic plasticity in chemoresistant gastric cancer during nutrient stress.

Molecular classification of gastric cancer (GC) identified a subgroup of patients showing chemoresistance and poor prognosis, termed SEM (Stem-like/Epithelial-to-mesenchymal transition/Mesenchymal) type in this study. Here, we show that SEM-type GC exhibits a distinct metabolic profile characterized by high glutaminase (GLS) levels. Unexpectedly, SEM-type GC cells are resistant to glutaminolysis inhibition. We show that under glutamine starvation, SEM-type GC cells up-regulate the 3 phosphoglycerate dehydrogenase (PHGDH)-mediated mitochondrial folate cycle pathway to produce NADPH as a reactive oxygen species scavenger for survival. This metabolic plasticity is associated with globally open chromatin structure in SEM-type GC cells, with ATF4/CEBPB identified as transcriptional drivers of the PHGDH-driven salvage pathway. Single-nucleus transcriptome analysis of patient-derived SEM-type GC organoids revealed intratumoral heterogeneity, with stemness-high subpopulations displaying high GLS expression, a resistance to GLS inhibition, and ATF4/CEBPB activation. Notably, coinhibition of GLS and PHGDH successfully eliminated stemness-high cancer cells. Together, these results provide insight into the metabolic plasticity of aggressive GC cells and suggest a treatment strategy for chemoresistant GC patients.

[Study of high selenium interfering with glucose and one-carbon metabolism in hepatocytes in vitro].

OBJECTIVE: To investigate the effects of high selenium environment on the expression of selenoproteins and enzymes related to glucose and one-carbon metabolism in normal human hepatocytes. METHODS: Ten different concentrations of selenomethionine(SeMet, 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 µmol/L) was added into the normal human hepatocyts and incubated for 48 hours. The expressions of selenoprotein(GPX1 and SELENOP1) and metabolic enzymes(PHGDH, SHMT1, MTHFR and MS) were analyzed by Western blot. RESULTS: When the concentration of SeMet was 0-10 µmol/L, the expression trend of selenoprotein(GPX1 and SELENOP1) is similar, which first increases and then decreases. There is a slight difference between the inflection points of GPX1 and SELENOP1, which are respectively 0.5 µmol/L and 0.1 µmol/L. The expression trend of serine de novo synthesis pathway key enzymes(PHGDH) and folate cycle metabolizing enzymes(SHMT1, MTHFR and MS) is similar to that of selenoproteins, which also increases first and then decreases, but the inflection points are different, which are respectively 0.1 µmol/L(PHGDH and SHMT1) and 0.01 µmol/L(MTHFR and MS). CONCLUSION: Under the high selenium environment, the glycolytic bypass-serine de novo synthesis pathway is activated to synthesize endogenous serine due to the insufficient intracellular serine supply, causing abnormal glucose metabolism, which is an important extension to the hypothesis of the molecular mechanism of high selenium causing IR.

Multi-omics Analysis of the Role of PHGDH in Colon Cancer.

Objectives: Serine metabolism is essential for tumor cells. Endogenous serine arises from de novo synthesis pathways. As the rate-limiting enzyme of this pathway, PHGDH is highly expressed in a variety of tumors including colon cancer. Therefore, targeted inhibition of PHGDH is an important strategy for anti-tumor therapy research. However, the specific gene expression and metabolic pathways regulated by PHGDH in colon cancer are still unclear. Our study was aimed to clarified the role of PHGDH in serine metabolism in colon cancer to provide new knowledge for in-depth understanding of serine metabolism and PHGDH function in colon cancer. Methods: In this study, we analyzed the gene expression and metabolic remodeling process of colon cancer cells (SW620) after targeted inhibition of PHGDH by gene transcriptomics and metabolomics. LC-MS analysis was performed in 293T cells to PHGDH gene transcription and protein post-translational modification under depriving exogenous serine. Results: We found that amino acid transporters, amino acid metabolism, lipid synthesis related pathways compensation and other processes are involved in the response process after PHGDH inhibition. And ATF4 mediated the transcriptional expression of PHGDH under exogenous serine deficiency conditions. While LC-MS analysis of post-translational modification revealed that PHGDH produced changes in acetylation sites after serine deprivation that the K289 site was lost, and a new acetylation site K21was produced. Conclusion: Our study performed transcriptomic and metabolomic analysis by inhibiting PHGDH, thus clarifying the role of PHGDH in gene transcription and metabolism in colon cancer cells. The mechanism of high PHGDH expression in colon cancer cells and the acetylation modification that occurs in PHGDH protein were also clarified by serine deprivation. In our study, the role of PHGDH in serine metabolism in colon cancer was clarified by multi-omics analysis to provide new knowledge for in-depth understanding of serine metabolism and PHGDH function in colon cancer.

The Global mRNA Expression Profiles of Inhibiting PHGDH Induced Cisplatin Resistance in Gastric Cancer.

Objective: Drug resistance is the main hindrance to improve the prognosis of patients with gastric cancer. Amino
acid metabolic reprograming is essential to satisfy the different requirements of cancer cells during drug resistance,
of which serine deprivation could promote resistance to cisplatin in gastric cancer. As the key enzyme in the de novo
biosynthesis of serine, phosphoglycerate dehydrogenase (PHGDH) inhibition could also induce cisplatin resistance in
gastric cancer. This study aims to reveal the potential mechanisms of drug resistance induced by PHGDH inhibition via
exploring the global mRNA expression profiles.
Materials and Methods: In this experimental study, the viability and the apoptotic rate of gastric cancer cells
were evaluated by using Cell Counting Kit-8 (CCK-8) analysis and flow cytometric determination, respectively. The
identification of differentially expressed genes (DEGs) was tested by mRNA-sequencing (mRNA-Seq) analysis. The
confirmation of sequencing results was verified using real-time quantitative reverse transcription polymerase chain
reaction (RT-qPCR).
Results: The inhibition of PHGDH significantly increased the viability and decreased the apoptotic rate induced by cisplatin
in gastric cancer cells. mRNA-Seq analysis revealed that the combined treatment of NCT503 reduced the number of DEGs
induced by cisplatin. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment
Analysis (GSEA) showed that unfolded protein response, ECM receptor interaction and cell cycle signaling pathways were
modulated by NCT503 treatment. Hub genes were identified by using protein-protein interaction network modeling, of which E1A binding protein p300 (EP300) and heat shock protein family A (Hsp70) member 8 (HSPA8) act as the vital genes in cisplatin resistance induced by the inhibition of PHGDH.
Conclusion: These findings suggested that the inhibition of PHGDH promoted cisplatin resistance in gastric cancer
through various intercellular mechanisms. And appropriate serine supplementation or the modulation of EP300 and
HSPA8 may be of great help in overcoming cisplatin resistance in gastric cancer.

Revealing a New Family of D-2-Hydroxyglutarate Dehydrogenases in Escherichia coli and Pantoea ananatis Encoded by ydiJ.

In E. coli and P. ananatis, L-serine biosynthesis is initiated by the action of D-3-phosphoglycerate dehydrogenase (SerA), which converts D-3-phosphoglycerate into 3-phosphohydroxypyruvate. SerA can concomitantly catalyze the production of D-2-hydroxyglutarate (D-2-HGA) from 2-ketoglutarate by oxidizing NADH to NAD+. Several bacterial D-2-hydroxyglutarate dehydrogenases (D2HGDHs) have recently been identified, which convert D-2-HGA back to 2-ketoglutarate. However, knowledge about the enzymes that can metabolize D-2-HGA is lacking in bacteria belonging to the Enterobacteriaceae family. We found that ydiJ encodes novel D2HGDHs in P. ananatis and E. coli, which were assigned as D2HGDHPa and D2HGDHEc, respectively. Inactivation of ydiJ in P. ananatis and E. coli led to the significant accumulation of D-2-HGA. Recombinant D2HGDHEc and D2HGDHPa were purified to homogeneity and characterized. D2HGDHEc and D2HGDHPa are homotetrameric with a subunit molecular mass of 110 kDa. The pH optimum was 7.5 for D2HGDHPa and 8.0 for D2HGDHEc. The Km for D-2-HGA was 208 µM for D2HGDHPa and 83 µM for D2HGDHEc. The enzymes have strict substrate specificity towards D-2-HGA and displayed maximal activity at 45 °C. Their activity was completely inhibited by 0.5 mM Mn2+, Ni2+ or Co2+. The discovery of a novel family of D2HGDHs may provide fundamental information for the metabolic engineering of microbial chassis with desired properties.

The involvement of Parkin-dependent mitophagy in the anti-cancer activity of Ginsenoside.

Colon cancer, the third most frequent occurred cancer, has high mortality and extremely poor prognosis. Ginsenoside, the active components of traditional Chinese herbal medicine Panax ginseng, exerts antitumor effect in various cancers, including colon cancer. However, the detailed molecular mechanism of Ginsenoside in the tumor suppression have not been fully elucidated. Here, we chose the representative ginsenoside Rg3 and reported for the first time that Rg3 induces mitophagy in human colon cancer cells, which is responsible for its anticancer effect. Rg3 treatment leads to mitochondria damage and the formation of mitophagosome; when autophagy is inhibited, the clearance of damaged mitochondria can be reversed. Next, our results showed that Rg3 treatment activates the PINK1-Parkin signaling pathway and recruits Parkin and ubiquitin proteins to mitochondria to induce mitophagy. GO analysis of Parkin targets showed that Parkin interacts with a large number of mitochondrial proteins and regulates the molecular function of mitochondria. The cellular energy metabolism enzyme GAPDH is validated as a novel substrate of Parkin, which is ubiquitinated by Parkin. Moreover, GAPDH participates in the Rg3-induced mitophagy and regulates the translocation of Parkin to mitochondria. Functionally, Rg3 exerts the inhibitory effect through regulating the nonglycolytic activity of GAPDH, which could be associated with the cellular oxidative stress. Thus, our results revealed GAPDH ubiquitination by Parkin as a crucial mechanism for mitophagy induction that contributes to the tumor-suppressive function of ginsenoside, which could be a novel treatment strategy for colon cancer.

Identification of a novel PHGDH covalent inhibitor by chemical proteomics and phenotypic profiling.

The first rate-limiting enzyme of the serine synthesis pathway (SSP), phosphoglycerate dehydrogenase (PHGDH), is hyperactive in multiple tumors, which leads to the activation of SSP and promotes tumorigenesis. However, only a few inhibitors of PHGDH have been discovered to date, especially the covalent inhibitors of PHGDH. Here, we identified withangulatin A (WA), a natural small molecule, as a novel covalent inhibitor of PHGDH. Affinity-based protein profiling identified that WA could directly bind to PHGDH and inactivate the enzyme activity of PHGDH. Biolayer interferometry and LC-MS/MS analysis further demonstrated the selective covalent binding of WA to the cysteine 295 residue (Cys295) of PHGDH. With the covalent modification of Cys295, WA blocked the substrate-binding domain (SBD) of PHGDH and exerted an allosteric effect to induce PHGDH inactivation. Further studies revealed that with the inhibition of PHGDH mediated by WA, the glutathione synthesis was decreased and intracellular levels of reactive oxygen species (ROS) were elevated, leading to the inhibition of tumor proliferation. This study indicates WA as a novel PHGDH covalent inhibitor, which identifies Cys295 as a novel allosteric regulatory site of PHGDH and holds great potential in developing anti-tumor agents for targeting PHGDH.

Vitamin C promotes anti-leukemia of DZNep in acute myeloid leukemia.

The epigenetic treatment by 3-Deazaneplanocin A (DZNep), a histone methyltransferase inhibitor, shows great potential against acute myeloid leukemia (AML). However, the variant sensitivity and incomplete response to DZNep are commonly observed. Here, we reveal that vitamin C (Vc) dramatically promotes DZNep response against leukemic cells in different cell lines and primary AML samples. Vc enhances apoptosis and differentiation induced by DZNep in different AML cell lines in vitro and reduces leukemia progression in vivo. At the molecular level, Vc downregulates an enzyme of serine synthesis named D-3-phosphoglycerate dehydrogenase (PHGDH), as well as BCL2, an anti-apoptotic gene. Over-expression of PHGDH reverses the Vc-enhanced anti-leukemic effect of DZNep in AML cells. Therefore, our findings provide an effective approach to reduce the resistance against epigenetic treatment by Vc, which shows a potential improvement of their combination in AML patients.

Impact of serine and serine synthesis genes on H2S release in Saccharomyces cerevisiae during wine fermentation.

Excessive hydrogen sulfide (H2S) during fermentation causes undesirable sensory properties in wine. In yeast, serine functions as a precursor in the biosynthesis of S-containing compounds, which facilitates H2S consumption. To investigate the effect of serine on H2S release and the underlying mechanism, extracellular and intracellular serine levels were separately increased under fermentation conditions. The results show that, although the abundance of extracellular serine was ineffective in decreasing H2S levels, increased levels of intracellular serine levels from SER1 and SER2 overexpression reduced H2S release through increased consumption of sulfur metabolites. In contrast, SER33 overexpression significantly increased H2S release, and the metabolites and gene expression profile of the sulfur assimilation pathway indicates that SER33 regulated MET17, which mediated H2S release. Our study revealed valuable insights on the relationship between serine levels and H2S release, and may be helpful in understanding the H2S regulation mechanism in yeast during fermentation.

1-Deoxysphinganine initiates adaptive responses to serine and glycine starvation in cancer cells via proteolysis of sphingosine kinase.

Cancer cells may depend on exogenous serine, depletion of which results in slower growth and activation of adaptive metabolic changes. We previously demonstrated that serine and glycine (SG) deprivation causes loss of sphingosine kinase 1 (SK1) in cancer cells, thereby increasing the levels of its lipid substrate, sphingosine (Sph), which mediates several adaptive biological responses. However, the signaling molecules regulating SK1 and Sph levels in response to SG deprivation have yet to be defined. Here, we identify 1-deoxysphinganine (dSA), a noncanonical sphingoid base generated in the absence of serine from the alternative condensation of alanine and palmitoyl CoA by serine palmitoyl transferase, as a proximal mediator of SG deprivation in SK1 loss and Sph level elevation upon SG deprivation in cancer cells. SG starvation increased dSA levels in vitro and in vivo and in turn induced SK1 degradation through a serine palmitoyl transferase-dependent mechanism, thereby increasing Sph levels. Addition of exogenous dSA caused a moderate increase in intracellular reactive oxygen species, which in turn decreased pyruvate kinase PKM2 activity while increasing phosphoglycerate dehydrogenase levels, and thereby promoted serine synthesis. We further showed that increased dSA induces the adaptive cellular and metabolic functions in the response of cells to decreased availability of serine likely by increasing Sph levels. Thus, we conclude that dSA functions as an initial sensor of serine loss, SK1 functions as its direct target, and Sph functions as a downstream effector of cellular and metabolic adaptations. These studies define a previously unrecognized "physiological" nontoxic function for dSA.

Reversing malignant biological behavior of osteosarcoma by knocking down 3-phosphoglycerin dehydrogenase targeting energy metabolism / 解剖学报

Objective To investigate the effect of knock⁃down 3⁃phosphoglycerin dehydrogenase (PHGDH) targeting energy metabolism on malignant biological behavior and osteogenic differentiation of human osteosarcoma 143B cells. Methods Real⁃time PCR and Western blotting were used to detect the expression of PHGDH in osteoblasts hFOB1. 19 and osteosarcoma cells TE85, MG63 and 143B with different malignant degrees. The short hairpin RNA (shRNA)⁃PHGDH recombinant plasmid was transfected into 143 B cells by liposome transfection method. The expression of PHGDH was detected by Real⁃time PCR and Western blotting. Crystal violet staining, cell counting and CCK⁃8 assay were used to detect cell proliferation; wound healing assay was used to detect cell parallel migration ability, and Transwell assay was used to detect cell vertical migration and invasion ability. Annexin V⁃FITC/ PI double staining and DAPI staining were used to detect apoptosis; Alkaline phosphatase(ALP) staining and alizarin red S staining were used to detect osteogenic differentiation. Western blotting was used to detect the expression of Runt related transcription factor 2 (Runx2) and osteocalcin (OC) . The expression of genes related to energy metabolism, glucose transporter⁃1 (GLUT1), 6⁃ phosphofructokinase⁃1(PFK1), pyruvate kinae subtype M2 (PKM2), lactate dehydrogenase A (LDHA) was detected by Real⁃time PCR. Lactic acid secretion was detected by lactic acid detection kit. Adenosine triphosphate(ATP) production was detected by ATP detection kit. Results The expression of PHGDH in 143B cells was significantly higher than that in hFOB1. 19, MG63 and TE85 cells (P < 0. 01) . After the transfection of shRNA⁃PHGDH recombinant plasmid, the expression of PHGDH in 143 B cells decreased (P<0. 01), proliferation ability decreased (P<0. 01), cell migration and invasion ability decreased (P < 0. 01), apoptosis rate increased (P < 0. 01), ALP staining positive rate increased (P < 0. 01), alizarin red staining positive rate increased (P < 0. 05), Runx2 (P < 0. 05) and OC expression increased (P < 0. 01), expression of genes related to energy metabolism (GLUT1, PFK1, PKM2, LDHA) decreased (P < 0. 01), lactic acid decreased (P < 0. 01), ATP increased (P < 0. 05) . Conclusion Knocking down of PHGDH can inhibit the proliferation, migration and invasion of human osteosarcoma 143B cells through energy metabolism, promote their apoptosis and promote their osteogenic differentiation.

Expressions and roles of key enzymes in serine synthesis pathway in NaAsO2-treated HaCaT cells / 环境与职业医学

Background The key enzymes of serine synthesis pathway (SSP) play an important role in tumor growth, proliferation, and invasion, but their roles in arsenic carcinogenesis are unclear. Objective To observe the effects of NaAsO2 treatment on the expressions of key enzymes [such as phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH)] of SSP and on the ability to proliferate and migrate in human immortalized skin keratinocytes (HaCaT) and NaAsO2-induced malignantly transformed HaCaT (T-HaCaT), and to explore the roles of SSP key enzymes in arsenic carcinogenesis. Methods (1) The T-HaCaT cells constructed earlier by our research team were divided into a passage control (0 μmol·L−1 NaAsO2) group, a T-HaCaT (0.5 μmol·L−1 NaAsO2) group, a NCT503 (PHGDH inhibitor, 25 μmol·L−1) group, and a NCT503 (25 μmol·L−1) + T-HaCaT (0.5 μmol·L−1 NaAsO2) group. Western blotting was used to detect the protein expression levels of SSP key enzymes in the passage control group and the T-HaCaT group. CCK8 assay and cell scratch test were used to detect the proliferation and migration rates of cells in each group respectively. (2) Well-grown logarithmic-phase HaCaT cells were treated with 0, 0.625, 1.25, and 2.5 μmol·L−1 NaAsO2 for 0, 24, 48, and 72 h to detect cell proliferation rate and protein expression levels of SSP key enzymes. In the subsequent experiment, HaCaT cells were pretreated with 25 μmol·L−1 NCT503 for 6 h, and then treated with 2.5 μmol·L−1 NaAsO2 for 72 h continuously. The experimental groups included a control (0 μmol·L−1 NaAsO2) group, an exposure (2.5 μmol·L−1 NaAsO2) group, a pretreatment (25 μmol·L−1 NCT503) group, and a pretreatment (25 μmol·L−1 NCT503) + exposure (2.5 μmol·L−1 NaAsO2) group, to detect the proliferation rate of cells in each group. Results The protein expression level of PHGDH in the T-HaCaT group were 1.60 times higher than that in the passage control group (P<0.05), and its proliferation rate (177.51%±14.69%) and migration rate (53.85%±0.94%) were also higher than the passage control group’s (100.00%±0.00% and 24.30%±2.26%) (both Ps<0.05), respectively. After the NCT503 intervention, the proliferation rate (144.97%±8.08%) and migration rate (35.80%±0.99%) of cells in the NCT503 + T-HaCaT group were lower than those in the T-HaCaT group (both P<0.05). The proliferation rate of HaCaT cells after NaAsO2 exposure for 72 h increased with the increase of exposure concentration (r=0.862, P<0.05), and consistently, the protein levels of SSP key enzymes in HaCaT cells in each exposure group were higher than those in the control group (all P<0.05). The proliferation rate of HaCaT cells treated with 2.5 μmol·L−1 NaAsO2 increased with the extension of exposure time (r=0.775, P<0.05), which was consistent with the changes of PHGDH levels in cells. After the NCT503 intervention, the proliferation rate of the pretreatment + exposure group was significantly lower than that of the exposure group (P<0.05). Conclusion The key enzymes of SSP may play an important role in the proliferation of T-HaCaT cells induced by NaAsO2.

Discovery of novel inhibitors of human phosphoglycerate dehydrogenase by activity-directed combinatorial chemical synthesis strategy.

Serine, the source of the one-carbon units essential for de novo purine and deoxythymidine synthesis plays a crucial role in the growth of cancer cells. Phosphoglycerate dehydrogenase (PHGDH) which catalyzes the first, rate-limiting step in de novo serine biosynthesis has become a promising target for the cancer treatment. Here we identified H-G6 as a potential PHGDH inhibitor from the screening of an in-house small molecule library based on the enzymatic assay. We adopted activity-directed combinatorial chemical synthesis strategy to optimize this hit compound. Compound b36 was found to be the noncompetitive and the most promising one with IC50 values of 5.96 ± 0.61 µM against PHGDH. Compound b36 inhibited the proliferation of human breast cancer and ovarian cancer cells, reduced intracellular serine synthesis, damaged DNA synthesis, and induced cell cycle arrest. Collectively, our results suggest that b36 is a novel PHGDH inhibitor, which could be a promising modulator to reprogram the serine synthesis pathway and might be a potential anticancer lead worth further exploration.