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.

Construction and characterization of Escherichia coli D-3-phosphoglycerate dehydrogenase mutants with feedback-inhibition relief / 生物工程学报

3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) is the key enzyme in L-serine biosynthesis and its coding gene is serA. PGDH is feedback inhibited by L-serine. In order to relieve the feedback-inhibition of PGDH by L-serine, H344 or D346 or D364 were chosen for site directed mutagenesis. The mutants were generated by the standard QuikChange mutagenesis, further subcloned into expression vector pT7-7 and transformed into Escherichia coli BL21 (DE3) cells. The recombinant cells were collected after cultured in LB media post induced by isopropyl beta-Dthiogalactopyranoside. The enzymes were purified by anion exchange chromatography, and SDS-PAGE showed that the purified enzymes were homogenous. Enzyme characterization indicated that the mutant enzyme showed similar activity, optimal temperature, and optimal pH as that of the wild-type enzyme. Moreover, feedback inhibition study showed that the activity of the double mutant (N346A/H344A) could remain 96% in the presence of serine up to 160 mmol/L, whereas the activity of the wild-type enzyme remains only 50% in the presents of serine of 7 μmol/L, thus successfully relieving the feedback inhibition of PGDH with its activity remained.

Phosphoglycerate dehydrogenase and tumors / 国际儿科学杂志

Phosphoglycerate dehydrogenase (phosphoglycerate dehydrogenase,PHGDH) encodes 3-phosphoglycerate dehydrogenase,the first enzyme branching from glycolysis in the serine biosynthetic pathway.PHGDH uses NAD as a cofactor to oxidizes glycolytic intermediate,3-phosphoglycerate,into phosphor-hydroxypyruvate,which then convert into serine by a series of enzymes in the pathway.Serine is essential for synthesis of proteins and other biomolecules needed for cell proliferation,including nucleotides,phosphatidyl-serine,and sphingosine.Recent studies found that subsets of human malignant cancers have high levels of PHGDH,a key enzyme for serine biosynthesis,and these cancer cells are dependent on PHGDH for their growth and survival.In this paper we made a review on PHGDH gene about its construction,function and relation with tumors.