Category Archives: RESEARCH ARTICLE

Biochemical and cellular studies of three human 3‐phosphoglycerate dehydrogenase variants responsible for pathological reduced L‐serine levels

Posted on 31 August 2023 by

In the brain, L-serine is produced through the phosphorylated pathway (PP). hPHGDH catalyzes the first and rate-limiting step in the PP. Three variants related to hPHGDH deficiency and Neu-Laxova syndrome have been studied. V261M, V425M, and V490M substitutions alter the kinetic and structural properties of hPHGDH. Variants ectopic expression results in protein aggregation and reduced L-serine level.

Abstract

In the brain, the non-essential amino acid L-serine is produced through the phosphorylated pathway (PP) starting from the glycolytic intermediate 3-phosphoglycerate: among the different roles played by this amino acid, it can be converted into D-serine and glycine, the two main co-agonists of NMDA receptors. In humans, the enzymes of the PP, namely phosphoglycerate dehydrogenase (hPHGDH, which catalyzes the first and rate-limiting step of this pathway), 3-phosphoserine aminotransferase, and 3-phosphoserine phosphatase are likely organized in the cytosol as a metabolic assembly (a “serinosome”). The hPHGDH deficiency is a pathological condition biochemically characterized by reduced levels of L-serine in plasma and cerebrospinal fluid and clinically identified by severe neurological impairment. Here, three single-point variants responsible for hPHGDH deficiency and Neu-Laxova syndrome have been studied. Their biochemical characterization shows that V261M, V425M, and V490M substitutions alter either the kinetic (both maximal activity and K _m for 3-phosphoglycerate in the physiological direction) and the structural properties (secondary, tertiary, and quaternary structure, favoring aggregation) of hPHGDH. All the three variants have been successfully ectopically expressed in U251 cells, thus the pathological effect is not due to hindered expression level. At the cellular level, mistargeting and aggregation phenomena have been observed in cells transiently expressing the pathological protein variants, as well as a reduced L-serine cellular level. Previous studies demonstrated that the pharmacological supplementation of L-serine in hPHGDH deficiencies could ameliorate some of the related symptoms: our results now suggest the use of additional and alternative therapeutic approaches.

Downregulation of microRNA‐326 enhances ZNF322A expression, transcriptional activity and tumorigenic effects in lung cancer

Posted on 30 August 2023 by

Oncogenic ZNF322A transcription factor is overexpressed in lung cancer. Downregulated miR-326 promotes ZNF322A-induced tumor growth and metastasis. This study reveals that miR-326-low/ZN322A-high profile is a biomarker to predict poor prognosis in lung cancer.

Abstract

Zinc finger protein ZNF322A is an oncogenic transcription factor. Overexpression of ZNF322A activates pro-metastasis, cancer stemness, and neo-angiogenesis-related genes to enhance lung cancer progression. However, the upstream regulator of ZNF322A is not well defined. Dysregulation of microRNAs (miRNAs) can mediate cancer cell growth, migration, and invasion to promote tumorigenesis. Here, we uncover the mechanism of miRNA-mediated transcriptional regulation in ZNF322A-driven oncogenic events. ZNF322A harbors several putative miRNA-binding sites in the 3′-untranslated region (UTR). We validated that miR-326 downregulated ZNF322A-3′-UTR luciferase activity and mRNA expression. Furthermore, miR-326 suppressed the expression of ZNF322A-driven cancer-associated genes such as cyclin D1 and alpha-adducin. Reconstitution experiments by ectopic overexpression of ZNF322A abolished miR-326-suppressed cancer cell proliferation and cell migration capacity. Moreover, miR-326 attenuated ZNF322A-induced tumor growth and lung tumor metastasis in vivo. Clinically, the expression of miR-326 negatively correlated with ZNF322A mRNA expression in surgically resected tissues from 120 non-small cell lung cancer (NSCLC) patients. Multivariate Cox regression analysis demonstrated that NSCLC patients with low miR-326/high ZNF322A profile showed poor overall survival. Our results reveal that the deregulated expression of miR-326 leads to hyperactivation of ZNF322A-driven oncogenic signaling. Targeting the miR-326/ZNF322A axis would provide new therapeutic strategies for lung cancer patients.

Reduced iron and cobalt levels in response to curcumin supplementation are not responsible for the prolonged larval development and do not affect the oxidative stress tolerance and polyamine status of D. melanogaster

Posted on 19 August 2023 by

Curcumin supplementation, similar to the iron chelator bathophenanthroline, lowered the iron but also the cobalt content, and to a lesser extent affected the manganese and zinc status of D. melanogaster. However, these alterations in trace metal balance did not affect catalase activity, oxidative stress tolerance, and polyamine status in fruit flies. In contrast, in curcumin-supplemented mice, the spleen exhibits an elevated spermidine production, which is most probably related to a compensatory growth due to curcumin-induced iron deficiency.

Abstract

Recent reports indicated that the phytochemical curcumin possesses iron-chelating activity. Here, by employing the fruit fly Drosophila melanogaster, we conducted feeding studies supplementing curcumin or, as a control, the iron chelator bathophenanthroline (BPA). First, the absorption and further metabolization of dietary curcuminoids were proved by metabolomics analyses. Next, we found that 0.2% dietary curcumin, similar to BPA, lowered the iron but also the cobalt content, and to a lesser extent affected the manganese and zinc status. Supplementation during larval stages was required and sufficient for both compounds to elicit these alterations in adult animals. However, curcumin-induced retarded larval development was not attributable to the changed trace metal status. In addition, a reduction in the iron content of up to 70% by curcumin or BPA supplementation did not reduce heme-dependent catalase activity and tolerance toward H₂O₂ in D. melanogaster. Moreover, polyamines were not influenced by curcumin treatment and decreased iron levels. This was confirmed for selected organs from 0.2% curcumin-treated mice, except for the spleen. Here, elevated spermidine level and concomitant upregulation of genes involved in polyamine production were associated with a putatively anemia-derived increased spleen mass. Our data underline that the metal-chelating property of curcumin needs to be considered in feeding studies.

Dapagliflozin improves diabetic cognitive impairment via indirectly modulating the mitochondria homeostasis of hippocampus in diabetic mice

Posted on 19 August 2023 by

The goal of this study is to determine the underlying mechanism and look into dapagliflozin's impact on diabetic cognitive impairment. Diabetes-induced cognitive dysfunction was attenuated by dapagliflozin and the effect was indirect rather than direct.

Abstract

Cognitive impairment is increasingly recognized as an important comorbidity of diabetes progression; however, the underlying molecular mechanism is unclear. Dapagliflozin, an inhibitor of sodium-glucose co-transporter 2 (SGLT2), has shown promising effects against diabetes in rodent experiments and human clinical assays. This study aimed to determine the underlying mechanism and examine the effect of dapagliflozin on diabetic cognitive impairment. To create an in vivo model of diabetic cognitive impairment, streptozotocin (STZ)-induced diabetic mice were used. Dapagliflozin was administered to mice for 8 weeks. The context fear condition and Morris water maze test was used to evaluate mice's behavioral change. Western blotting was used to evaluate protein expression. Hematoxylin and eosin (HE) and Nissl staining were applied to monitor morphological and structural changes. Congo red staining was performed to identify the formation of senile plaques. Mitochondria morphology was examined using a transmission electron microscope, and blood flow in the mouse cerebral cortex was measured using a laser Doppler imaging assay. Comparison to the diabetes mellitus (DM) group, the dapagliflozin group had lower glucose levels. Behavioral studies have shown that dapagliflozin can restore memory deficits in diabetic mice. The murky cell membrane edges and Nissl bodies more difficult to identify in the DM group were revealed by HE and Nissl staining, which were both improved by dapagliflozin treatment. Dapagliflozin inhibited the progression of Aβ generation and the reduced cerebral blood flow in the DM group was rescued. After dapagliflozin treatment, damaged mitochondria and lack of SGLT2 in the hippocampus and cortex of diabetic mice were repaired. Diabetes-induced cognitive dysfunction was attenuated by dapagliflozin and the effect was indirect rather than direct.

Bioactive peptides in preterm human milk: Impact of maternal characteristics and their association to neonatal outcomes

Posted on 16 August 2023 by

Hormone content of very preterm human milk is partially determined by maternal factors most of them regarding metabolic health and obstetric characteristics. Human milk concentration impacts on growth and development of preterm infants during their stay in the neonatal unit.

Abstract

Human milk adipokines in term babies seem partially determined by maternal factors and affect infant's development. We aimed to describe bioactive peptide concentration in very preterm human milk and associations to maternal characteristics and postnatal growth. Mothers delivering ≤32 weeks of gestation and their infant/s were recruited. At 4 weeks of lactation, an aliquot of 24-h-pooled milk was collected for exclusively breastfeeding dyads. Insulin, leptin, adiponectin, and milk fat globule epidermal growth factor-8 (MFG-E8) were measured by enzyme-linked immunoabsorbent assay in skimmed milk. One hundred mothers (28.8 ± 2.3 weeks at delivery) provided a milk sample. Milk insulin was related to gestational age, pre-pregnancy body mass index (BMI), and galactagogue treatment (final model: adjusted R ²: 0.330, p < 0.0001; adjusted β coefficients: galactagogue treatment: 0.348, p 0.001; pre-pregnancy BMI: 0.274, p 0.009; gestational age: −0.290, p 0.007). Adiponectin was higher in mothers with gestational diabetes (30.7 ± 6.5 vs. 24.8 ± 8 ng/mL, p 0.044). Leptin was associated with pre-pregnancy BMI (Spearman's ρ: 0.648, p < 0.0001) and MFG-E8 to presence of labor and multiple pregnancy (final linear regression model, R ²: 0.073, p 0.028, adjusted β coefficients: presence of labor −0.229, p 0.050; twins: −0.192, p 0.099). Milk adiponectin was associated with a greater decrease in length z-scores from birth to 28 days (Pearson's r: −0.225, p 0.032) and to discharge (Pearson's r: −0.290, p 0.003). Milk MFG-E8 was lower in milk of mothers whose babies experienced late-onset sepsis (13.3 ± 5.8 vs. 16.8 ± 6.3 μg/mL, p 0.023). Adipokines levels in preterm human milk are partially related to maternal metabolic status. Milk peptide concentration associates with early neonatal growth trajectories.

Caffeic acid phenethyl ester surmounts acquired resistance of AZD9291 in non‐small cell lung cancer cells

Posted on 9 August 2023 by nYaw Twum, nKent Marshall, nWeimin Gaon

We developed and characterized a new tyrosine kinase inhibitor resistant non-small cell lung cancer (NSCLC) line, HCC827GR, which (1) was significantly resistant to both gefitinib and AZD9291; (2) had more in vitro tumorigenic potential as evident by a larger colony size from the anchorage-independent growth assay; and (3) exhibited a total of 26 differentially expressed genes (≥two fold-change) as compared to HCC827. These genes were involved in regulating cell growth, transcription, phase 1 metabolism, cell cycle, and apoptosis. Additionally, AZD9291 in combination with CAPE partially reverted the AZD9291 resistance in HCC827GR cells. This was exhibited by (1) synergistically suppressed cell viability; (2) cell cycle arrest and apoptosis induced through suppressing EGFR activation and modulating p53, p21, cyclin D1, and survivin protein expressions; and (3) differentially regulating genes involved in cancer drug resistance pathways.

Abstract

Epithelial growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the first-line therapy for EGFR mutated non-small cell lung cancer (NSCLC); however, resistance rapidly develops. The objective of this study was therefore to establish and characterize a gefitinib resistant NSCLC line (HCC827GR) and evaluate the therapeutic effects of natural products in combination with third-generation EGFR-TKI, AZD9291. The IC₅₀ of gefitinib and AZD9291 in HCC827GR were significantly higher than those of HCC827 (p < 0.05). Furthermore, anchorage-independent colony assay indicated that HCC827GR cells were more aggressive than their predecessors. This was reflected by the gene/protein expression changes observed in HCC827GR versus HCC827 profiled by cancer drug resistance real-time polymerase chain reaction (RT-PCR) array and Western blot. Three natural products were screened and caffeic acid phenethyl ester (CAPE) exhibited the most significant combinative cytotoxic effect with AZD9291. Specifically, flow cytometry revealed that AZD9291 + CAPE considerably increased the fraction of cell in pre-G1 of the cell cycle and caspase-Glo3/7 assay showed a dramatic increase in apoptosis when compared to AZD9291 alone. Furthermore, Western blot showed significant downregulation of p-EGFR/p-AKT in HCC827GR cells treated with AZD9291 + CAPE as compared to AZD9291. Moreover, it is evident that AZD9291 + CAPE specifically resulted in a marked reduction in the protein expressions of the cell-proliferation-related genes p21, cyclin D1, and survivin. Finally, refined RT-PCR/Western blot data indicated that AZD9291 + CAPE may at least partially exert its synergistic effects via the PLK2 pathway. Together, these results suggest that CAPE is a clinically relevant compound to aid AZD9291 in treating EGFR-TKI resistant cells through modulating critical genes/proteins involved in cancer resistance/therapy.

Rat brown adipose tissue thermogenic markers are modulated by estrous cycle phases and short‐term fasting

Posted on 21 July 2023 by

This study confirmed that BAT exhibits morphological and functional changes in proestrus and diestrus. Moreover, BAT undergoes additional dynamic functional and morphological changes during short-term fasting.

Abstract

Brown adipose tissue (BAT) converts chemical energy into heat to maintain body temperature. Although fatty acids (FAs) represent a primary substrate for uncoupling protein 1 (UCP1)-dependent thermogenesis, BAT also utilizes glucose for the same purpose. Considering that estrous cycle effects on BAT are not greatly explored, we examined those of 6-h fasting on interscapular BAT (iBAT) thermogenic markers in proestrus and diestrus. We found that the percentage of multilocular adipocytes was lower in proestrus than in diestrus, although it was increased after fasting in both analyzed estrous cycle stages. Furthermore, the percentage of paucilocular adipocytes was increased by fasting, unlike the percentage of unilocular cells, which decreased in both analyzed stages of the estrous cycle. The UCP1 amount was lower in proestrus irrespectively of the examined dietary regimens. Regarding FA transporters, it was shown that iBAT CD36 content was increased in fasted rats in diestrus. In contrast to GLUT1, the level of GLUT4 was interactively modulated by selected estrous cycle phases and fasting. There was no change in insulin receptor and ERK1/2 activation, while AKT activation was interactively modulated by fasting and estrous cycle stages. Our study showed that iBAT exhibits morphological and functional changes in proestrus and diestrus. Moreover, iBAT undergoes additional dynamic functional and morphological changes during short-term fasting to modulate nutrient utilization and adjust energy expenditure.

Protein concentrations and activities of fatty acid desaturase and elongase enzymes in liver, brain, testicle, and kidney from mice: Substrate dependency

Posted on 20 July 2023 by

The liver had the highest capacity for PUFA biosynthesis, with limited activity in the brain, testicles, and kidney, while we failed to detect activity in the heart and lung. The protein content and activity of the enzymes were significantly correlated. The capacity for PUFA synthesis in mice mainly resides in the liver, with enzymes having preference for n-3 PUFAs.

Abstract

The synthesis rates of n-3 and n-6 polyunsaturated fatty acids (PUFAs) in rodents and humans are not agreed upon and depend on substrate availability independently of the capacity for synthesis. Therefore, we aimed to assess the activities of the enzymes for n-3 and n-6 PUFA synthesis pathways in liver, brain, testicle, kidney, heart, and lung, in relation to their protein concentration levels. Eight-week-old Balb/c mice (n = 8) were fed a standard chow diet (6.2% fat, 18.6% protein, and 44.2% carbohydrates) until 14 weeks of age, anesthetized with isoflurane and tissue samples were collected (previously perfused) and stored at −80°C. The protein concentration of the enzymes (Δ-6D, Δ-5D, Elovl2, and Elovl5) were assessed by ELISA kits; their activities were assayed using specific PUFA precursors and measuring the respective PUFA products as fatty acid methyl esters by gas chromatographic analysis. The liver had the highest capacity for PUFA biosynthesis, with limited activity in the brain, testicles, and kidney, while we failed to detect activity in the heart and lung. The protein concentration and activity of the enzymes were significantly correlated. Furthermore, Δ-6D, Δ-5D, and Elovl2 have a higher affinity for n-3 PUFA precursors compared to n-6 PUFA. The capacity for PUFA synthesis in mice mainly resides in the liver, with enzymes having preference for n-3 PUFAs.

Sodium Danshensu ameliorates cerebral ischemia/reperfusion injury by inhibiting CLIC4/NLRP3 inflammasome‐mediated endothelial cell pyroptosis

Posted on 17 July 2023 by

Mechanism of SDSS in inhibiting endothelial cell pyroptosis. In the priming step, NLRP3, pro-Caspase-1, GSDMD-full, pro-IL-1β, and pro-IL-18 were up-regulated. Furthermore, the translocation of CLIC4 from cytoplasm to the membrane induced chloride outflow, resulting in the assembly of NLRP3, ASC and Pro-Caspase-1 into a platform (activation step). By binding CLIC4 and blocking its membrane localization, SDSS inhibited chloride outflow, thus inhibiting the activation of NLRP3 inflammasome and then the cleavage of pro-Caspase-1 into Caspase-1. This inhibited pyroptosis along with the release of IL-1β and IL-18, resulting from Caspase-1-dependent GSDMD-N cleavage.

Abstract

Endothelial pyroptosis promotes cerebral ischemia/reperfusion injury (CIRI). Sodium Danshensu (SDSS) has been shown to attenuate CIRI and have anti-inflammatory properties in endothelial cells. However, the mechanism and effect of SDSS on alleviating endothelial pyroptosis after CIRI remains poorly understood. Thus, we aimed to investigate the efficacy and mechanism of SDSS in reducing endothelial pyroptosis. It has been shown that SDSS administration inhibited NLRP3 inflammasome-mediated pyroptosis. As demonstrated by protein microarrays, molecular docking, CETSA and ITDRF_CETSA, SDSS bound strongly to CLIC4. Furthermore, SDSS can decrease its expression and inhibit its translocation. Its effectiveness was lowered by CLIC4 overexpression but not by knockdown. Overall The beneficial effect of SDSS against CIRI in this study can be ascribed to blocking endothelial pyroptosis by binding to CLIC4 and then inhibiting chloride efflux-dependent NLRP3 inflammasome activation.

P38α contributes to TNF‐α‐induced IL‐8 production in human gingival cells

Posted on 14 July 2023 by nDan Mao, nHiroshi Inoue, nTakuya Notomi, nSeiji Godan

When phosphorylated at threonine 180 and tyrosine 182, p38α contributes to the induction of IL-8 by TNF-α in Ca9-22 cells. TNF-α-induced phosphorylation translocates NF-κB into the nucleus and then increases both IL-8 mRNA expression and secretion. Thus, p38α plays an important role in TNF-α-induced IL-8 production, providing a potential therapeutic target to prevent and treat periodontal disease.

Abstract

Tumor necrosis factor-alpha (TNF-α) is a major inflammatory cytokine that induces interleukin (IL)-8 production. Although some studies have reported the involvement of the p38 MAPK signaling pathway in TNF-α-induced IL-8 production, its specific regulatory mechanisms in gingival epithelial cells (GECs) are still poorly understood. In the present study, Ca9-22 cells were used as representative GECs to investigate the effect of p38 signaling on TNF-α-induced IL-8 production. We found that TNF-α enhanced IL-8 production in Ca9-22 cells by activating the p38 signaling pathway, and one of its isoforms, p38α, played a key role. P38α deletion markedly inhibited TNF-α-induced IL-8 expression in Ca9-22 cells, while p38α gene rescue could reverse this effect. Further studies revealed that TNF-α-induced IL-8 production was markedly reduced when the threonine 180 and tyrosine 182 p38α phosphorylation sites were targeted for mutagenesis to alanine and phenylalanine, respectively, suggesting their critical role in the process. In conclusion, p38α plays an important role in TNF-α-induced IL-8 production, providing a potential therapeutic target to prevent and treat periodontal disease.