HIF Pathway Research Articles

IDH1 Mutation Induces HIF-1α and Confers Angiogenic Properties in Chondrosarcoma JJ012 Cells.

Chondrosarcoma is a group of primary bone cancers that arise from transformed cells of chondrocytic lineage. Tumor recurrence and metastasis are devastating for patients with chondrosarcoma since there are no effective treatment options. IDH mutations occur in over 50% of tumors from patients with conventional or dedifferentiated chondrosarcomas and represent an attractive target for therapy. However, their role in the pathogenesis of chondrosarcoma remains largely unknown. In this study, we sought to determine the association of IDH mutation and HIF-1α in chondrosarcoma. We used the chondrosarcoma JJ012 cell line and its derived CRISPR/Cas9 mutant IDH1 (IDH1mut) knockout (KO) cells. RNA-Seq data analysis revealed downregulation of several HIF-1α target genes upon loss of IDH1mut. This was associated with reduced HIF-1α levels in the IDH1mut KO cells and tumors. Loss of IDH1mut also attenuated the expression of angiogenic markers in tumor tissues and abrogated the angiogenic capacity of JJ012 cells. Moreover, we observed that exogenous expression of HIF-1α significantly promoted anchorage-independent colony-formation by IDH1mut KO cells. These results suggest IDH1 mutation confers angiogenic and tumorigenic properties of JJ012 cells by inducing HIF-1α. Thus, the HIF pathway represents a promising candidate for combinatorial regimens to target IDH1 mutated chondrosarcomas.

Comprehensive Characterization of the Genomic Landscape in Chinese Pulmonary Neuroendocrine Tumors Reveals Prognostic and Therapeutic Markers (CSWOG-1901).

BackgroundPulmonary neuroendocrine tumors (pNETs) include typical carcinoid (TC), atypical carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC), and small cell lung carcinoma (SCLC). The optimal treatment strategy for each subtype remains elusive, partly due to the lack of comprehensive understanding of their molecular features. We aimed to explore differential genomic signatures in pNET subtypes and identify potential prognostic and therapeutic biomarkers.MethodsWe investigated genomic profiles of 57 LCNECs, 49 SCLCs, 18 TCs, and 24 ACs by sequencing tumor tissues with a 520-gene panel and explored the associations between genomic features and prognosis.ResultsBoth LCNEC and SCLC displayed higher mutation rates for TP53, PRKDC, SPTA1, NOTCH1, NOTCH2, and PTPRD than TC and AC. Small cell lung carcinoma harbored more frequent co-alterations in TP53-RB1, alterations in PIK3CA and SOX2, and mutations in HIF-1, VEGF and Notch pathways. Large cell neuroendocrine carcinoma (12.7 mutations/Mb) and SCLC (11.9 mutations/Mb) showed higher tumor mutational burdens than TC (2.4 mutations/Mb) and AC (7.1 mutations/Mb). 26.3% of LCNECs and 20.8% of ACs harbored alterations in classical non-small cell lung cancer driver genes. The presence of alterations in the homologous recombination pathway predicted longer progression-free survival in advanced LCNEC patients with systemic therapy (P = .005) and longer overall survival (OS) in SCLC patients with resection (P = .011). The presence of alterations in VEGF (P = .048) and estrogen (P = .018) signaling pathways both correlated with better OS in patients with resected SCLC.ConclusionWe performed a comprehensive genomic investigation on 4 pNET subtypes in the Chinese population. Our data revealed distinctive genomic signatures in subtypes and provided new insights into the prognostic and therapeutic stratification of pNETs.

Activation of PI3K/AKT Pathway Is a Potential Mechanism of Treatment Resistance in Small Cell Lung Cancer.

Here, we have investigated treatment resistance mechanisms in small cell lung cancer (SCLC) by focusing on comparing the genotype and phenotype in tumor samples of treatment-resistant and treatment-sensitive SCLC. We conducted whole-exome sequencing on paired tumor samples at diagnosis and relapse from 11 patients with limited-stage (LS)-SCLC and targeted sequencing of 1,021 cancer-related genes on cell-free DNA at baseline and paired relapsed samples from 9 additional patients with LS-SCLC. Furthermore, we performed label-free mass spectrometry-based proteomics on tumor samples from 28 chemo-resistant and 23 chemo-sensitive patients with extensive-stage (ES)-SCLC. The main findings were validated in vitro in chemo-sensitive versus chemo-resistant SCLC cell lines and analyses of transcriptomic data of SCLC cell lines from a public database. Genomic analyses demonstrated that at relapse of LS-SCLC, genes in the PI3K/AKT signaling pathway were enriched for acquired somatic mutations or high-frequency acquired copy-number variants. Pathway analysis on differentially upregulated proteins from ES-SCLC cohort revealed enrichment in the HIF-1 signaling pathway. Importantly, 7 of 62 PI3K/AKT pathway genes containing acquired somatic copy-number amplifications were enriched in HIF-1 pathway. Analyses of transcriptomic data of SCLC cell lines from public databases confirmed upregulation of PI3K/AKT and HIF-1 pathways in chemo-resistant SCLC cell lines. Furthermore, chemotherapy-resistant cell lines could be sensitive to PI3K inhibitors in vitro. PI3K/AKT pathway activation may be one potential mechanism underlying therapeutic resistance of SCLC. This finding warrants further investigation and provides a possible approach to reverse resistance to chemo/radiotherapy.

Abstract 109: Biological Pathways Enriched For Genes Associated With Lesion Burden In Cerebral Cavernous Malformation

Introduction: Familial Cerebral Cavernous Malformation (CCM) is an autosomal dominant disorder caused by mutations in KRIT1 , CCM2 and PDCD10 . CCMs can result in seizures, neurological deficits, and intracranial hemorrhage. Our prior research identified genetic modifiers that contribute to CCM progression. We hypothesized that genes associated with CCM progression (lesion burden, large lesion size, and hemorrhage) are enriched in biological pathways. We performed a functional enrichment analysis of top associated genes from a genome-wide panel to identify pathways that, when dysregulated, may contribute to CCM progression. Methods: Familial CCM cases (n=335) enrolled in the Brain Vascular Malformation Consortium were genotyped using Affymetrix Axiom Genome-Wide LAT1 Human Arrays. Logistic regression analysis was performed using an additive model, adjusting for age at enrollment and sex, to identify genetic variants associated with total lesion burden, large lesion burden (≥5mm), or hemorrhage. For nominally associated variants (P<0.001) mapping +/-5kb to genes, we performed an over-representation analysis using WebGestalt to identify enrichment of genes in KEGG biological pathways using a false discovery rate (FDR) < 0.15. Results: Our CCM GWAS study identified genes nominally associated with total lesion burden (n=298), large lesion burden (n=287), and hemorrhage (n=200). We found no significantly over-represented pathways for genes associated with large lesion burden and hemorrhage. However, six pathways were enriched for genes associated with total lesion burden, all with FDR=0.12: oxytocin signaling, synaptic vesicle cycle, HIF-1 signaling, neomycin, kanamycin and gentamicin biosynthesis, adrenergic signaling in cardiomyocytes, and gastric acid secretion. Interestingly, the HIF-1 pathway interacts with several pathways of interest in CCM, e.g., mTOR and PI3K-Akt. Conclusions: Genetic variants associated with total lesion burden in familial CCM map to genes that are likely enriched in KEGG biological pathways. These findings expand the list of candidate genetic modifiers for CCM lesion progression, including genes in the HIF-1 pathway, and necessitate further investigation in other independent CCM patient cohorts.

Network pharmacology integrated with experimental validation reveals the regulatory mechanism of action of Hehuan Yin decoction in polycystic ovary syndrome with insulin resistance

Ethnopharmacological relevanceHehuan Yin decoction (HHY), first recorded in the Jingyue Quanshu (published in 1624 A.D.), is composed of Albizia julibrissin Durazz. and Ampelopsis japonica (Thunb.) Makino. Aim of the studyThis study aimed to investigate the mechanism of action of HHY in treating polycystic ovary syndrome with insulin resistance (PCOS-IR). Materials and methodsNetwork pharmacology and molecular docking were used to predict active compounds, potential targets, and pathways for PCOS-IR treatment using HHY. Female Sprague–Dawley rats were administered letrozole (1 mg/kg) with a high-fat diet to establish a PCOS-IR model. Thereafter, symptoms, ovarian pathology, serum insulin resistance, and sex hormone levels were determined. Western blotting was used to determine the levels of PI3Kp85α, AKT, phospho (p)-AKT, and GSK3β in the ovaries of rats. ResultsNetwork pharmacology revealed 58 components in HHY and 182 potential targets that were shared between HHY and PCOS-IR. HHY could potentially treat PCOS-IR via the insulin resistance, PI3K/AKT, HIF-1, and steroid hormone biosynthesis pathways. Molecular docking revealed that PI3K, AKT1, GSK3β, IRS1, and EGFR had high affinities to HHY compounds. In the PCOS-IR rats, HHY significantly normalised the symptoms and ovarian pathology, increased follicle-stimulating hormone (FSH) and oestradiol levels in the serum, and decreased the levels of fasting plasma glucose and fasting insulin, as well as the insulin resistance index. HHY also decreased the luteinising hormone (LH) and testosterone levels and the LH/FSH ratio in the PCOS-IR rats and increased the levels of PI3K, p-AKT, and GSK3β in ovary tissue, which indicated the activation of the PI3K/AKT pathway. ConclusionsHHY can improve PCOS-IR symptoms via multiple pharmacological pathways and may be a potential alternative therapy for the treatment of PCOS-IR.

Structure-Activity Relationships and Transcriptomic Analysis of Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitors.

To evaluate the differences in action of commercially available 2-oxoglutarate mimetics and “branched-tail” oxyquinoline inhibitors of hypoxia-inducible factor prolyl hydroxylase (HIF PHD), the inhibitors’ IC50 values in the activation of HIF1 ODD-luciferase reporter were selected for comparative transcriptomics. Structure–activity relationship and computer modeling for the oxyquinoline series of inhibitors led to the identification of novel inhibitors, which were an order of magnitude more active in the reporter assay than roxadustat and vadadustat. Unexpectedly, 2-methyl-substitution in the oxyquinoline core of the best HIF PHD inhibitor was found to be active in the reporter assay and almost equally effective in the pretreatment paradigm of the oxygen-glucose deprivation in vitro model. Comparative transcriptomic analysis of the signaling pathways induced by HIF PHD inhibitors showed high potency of the two novel oxyquinoline inhibitors (#4896-3249 and #5704-0720) at 2 μM concentrations matching the effect of 30 μM roxadustat and 500 μM dimethyl oxalyl glycine in inducing HIF1 and HIF2-linked pathways. The two oxyquinoline inhibitors exerted the same activation of HIF-triggered glycolytic pathways but opposite effects on signaling pathways linked to alternative substrates of HIF PHD 1 and 3, such as p53, NF-κB, and ATF4. This finding can be interpreted as the specificity of the 2-methyl-substitute variant for HIF PHD2.

Identification of traditional Chinese drugs containing active ingredients for treating myocardial infarction and analysis of their therapeutic mechanisms by network pharmacology and molecular docking

To identify traditional Chinese drugs that contain active ingredients for treatment of myocardial infarction (MI) and explore their therapeutic mechanisms using network pharmacology and molecular docking technology. The TCMSP database was used for screening the traditional Chinese drugs containing active ingredients for treating MI, and the related targets of MI and the candidate drugs were obtained from Genecards, OMIM, PharmGkb and PharmMapper databases. The common target network of the drug targets and disease targets was established using Venny2.1.0 software. GO and KEGG signal pathway enrichment analysis of the common targets was performed, and the protein-protein interaction (PPI) network was constructed for the targets. The targets in the PPI network were analyzed to identify the key targets, for which GO and KEGG pathway enrichment analyses were performed. Molecular docking was performed for the candidate ingredients and the key targets, and a total score ≥6 was used as the criteria for screening the therapeutic ingredients and their docking binding with key targets was verified. A human umbilical vein endothelial cell (HUVEC) model of oxygen-glucose deprivation (OGD) was used to validate the candidate ingredients and the key therapeutic targets for MI by Western blotting. Our analysis identified Salvia miltiorrhiza and Dalbergiae odoriferae as the candidate drugs rich in active ingredients for treatment of MI. These ingredients involved 16 key therapeutic targets for MI, which participated in such biological processes as inflammatory response, angiogenesis, energy metabolism and oxidative stress and the pathways including HIF-1, VEGF, and TNF pathways. Sclareol and PTGS2 in Salvia miltiorrhiza and formononetin and KDR in Dalbergiae odoriferae all had high docking total scores. Western blotting showed that at medium and high doses, sclareol significantly inhibited PTGS2 expression and formononetin promoted KDR expressions in the cell models in a dose-dependent manner (P < 0.05). Both Salvia miltiorrhiza and Dalbergiae odoriferae have good therapeutic effects on MI. Sclareol in Salvia miltiorrhiza and formononetin in Dalbergiae odoriferae regulate the expressions of KDR and PTGS2, respectively, to modulate the inflammatory response, angiogenesis, oxidative stress and energy metabolism and thus produce myocardial protective effects.

Full-Length Transcriptome of Red Swamp Crayfish Hepatopancreas Reveals Candidate Genes in Hif-1 and Antioxidant Pathways in Response to Hypoxia-Reoxygenation.

Red swamp crayfish is particularly prone to exposure to hypoxia-reoxygenation stress on account of the respiration and rhythmic, light-dependent photosynthetic activity of the algae and aquatic grass. Up to now, the regulation mechanisms of the adverse effects of hypoxia-reoxygenation for this species were still unknown, especially the roles of the antioxidant enzymes in reducing oxidative damage during reoxygenation. To screen for vital genes or pathways upon hypoxic-reoxygenation stress, hepatopancreas gene expression profiles were investigated by using a strategy combining second and third generation sequencing. Five groups of samples, including hypoxia for 1 and 6 h with DO of 1.0 mg/L, reoxygenation for 1 and 12 h with DO of 6.8 mg/L, and the samples under normoxia condition, were used for transcriptome sequencing. Twenty Illumina cDNA libraries were prepared to screen for the differentially expressed genes (DEGs) among the 5 groups of samples. Based on the assembled reference full-length transcriptome, 389 and 533 significantly DEGs were identified in the groups under severe hypoxia treatment for 1 and 6 h, respectively. The top three enriched pathways for these DEGs were "protein processing in endoplasmic reticulum," "MAPK signaling pathway," and "endocytosis." Among these DEGs, hypoxia-inducible factor 1α (Hif-1α) and some Hif-1 downstream genes, such as Ugt-1, Egfr, Igfbp-1, Pk, and Hsp70, were significant differentially expressed when exposed to hypoxia stress. A series of antioxidant enzymes, including two types of superoxide dismutase (Cu/ZnSOD and MnSOD), catalase (CAT), and glutathione peroxidase (GPx), were identified to be differentially expressed during hypoxia-reoxygenation treatment, implying their distinct modulation roles on reoxygenation-induced oxidative stress. The full-length transcriptome and the critical genes characterized should contribute to the revelation of intrinsic molecular mechanism being associated with hypoxia/reoxygenation regulation and provide useful foundation for future genetic breeding of the red swamp crayfish.

Mechanistic Insight on Autophagy Modulated Molecular Pathways in Cerebral Ischemic Injury: From Preclinical to Clinical Perspective.

Cerebral ischemia is one of the most devastating brain injuries and a primary cause of acquired and persistent disability worldwide. Despite ongoing therapeutic interventions at both the experimental and clinical levels, options for stroke-related brain injury are still limited. Several evidence suggests that autophagy is triggered in response to cerebral ischemia, therefore targeting autophagy-related signaling pathways can provide a new direction for the therapeutic implications in the ischemic injury. Autophagy is a highly conserved lysosomal-dependent pathway that degrades and recycles damaged or non-essential cellular components to maintain neuronal homeostasis. But, whether autophagy activation promotes cell survival against ischemic injury or, on the contrary, causes neuronal death is still under debate. We performed an extensive literature search from PubMed, Bentham and Elsevier for various aspects related to molecular mechanisms and pathobiology involved in autophagy and several pre-clinical studies justifiable further in the clinical trials. Autophagy modulates various downstream molecular cascades, i.e., mTOR, NF-κB, HIF-1, PPAR-γ, MAPK, UPR, and ROS pathways in cerebral ischemic injury. In this review, the various approaches and their implementation in the translational research in ischemic injury into practices has been covered. It will assist researchers in finding a way to cross the unbridgeable chasm between the pre-clinical and clinical studies.

Prediction of the Mechanisms by Which Quercetin Enhances Cisplatin Action in Cervical Cancer: A Network Pharmacology Study and Experimental Validation.

Yimucao has been used as an herbal medicine to treat gynecological diseases. Common genes of Yimucao active compounds were investigated using network pharmacology. The components and targets of Yimucao were retrieved from the TCMSP database. Cervical cancer targets were collected from GeneCards, TTD, DisGeNET, and KEGG. Cisplatin-related genes were downloaded from GeneWeaver. The protein-protein interaction (PPI) network was created using the STRING database. A drug-bioactive compound-disease-target network was constructed using Cytoscape. GO and KEGG analyses were performed to investigate common targets of quercetin and cisplatin in cervical cancer. We found that quercetin was the highly bioactive compound in Yimucao. The drug-bioactive compound-disease-target network contained 93 nodes and 261 edges. Drug-related key targets were identified, including EGFR, IL6, CASP3, VEGFA, MYC, CCND1, ERBB2, FOS, PPARG, and CASP8. Core targets were primarily related to the response to metal ions, cellular response to xenobiotic stimulus, and transcription factor complex. The KEGG pathway analysis revealed that quercetin and cisplatin may affect cervical cancer through platinum drug resistance and the p53 and HIF-1 pathways. Furthermore, quercetin combined with cisplatin downregulated the expression of EGFR, MYC, CCND1, and ERBB2 proteins and upregulated CASP8 expression in HeLa and SiHa cells. Functionally, quercetin enhanced cisplatin-induced anticancer activity in cervical cancer cells. Our results indicate that quercetin can be used to overcome cisplatin resistance in cervical cancer cells.

Cavefish cope with environmental hypoxia by developing more erythrocytes and overexpression of hypoxia-inducible genes.

Dark caves lacking primary productivity can expose subterranean animals to hypoxia. We used the surface-dwelling (surface fish) and cave-dwelling (cavefish) morphs of Astyanax mexicanus as a model for understanding the mechanisms of hypoxia tolerance in the cave environment. Primitive hematopoiesis, which is restricted to the posterior lateral mesoderm in other teleosts, also occurs in the anterior lateral mesoderm in Astyanax, potentially pre-adapting surface fish for hypoxic cave colonization. Cavefish have enlarged both hematopoietic domains and develop more erythrocytes than surface fish, which are required for normal development in both morphs. Laboratory-induced hypoxia suppresses growth in surface fish but not in cavefish. Both morphs respond to hypoxia by overexpressing hypoxia-inducible factor 1 (hif1) pathway genes, and some hif1 genes are constitutively upregulated in normoxic cavefish to similar levels as in hypoxic surface fish. We conclude that cavefish cope with hypoxia by increasing erythrocyte development and constitutive hif1 gene overexpression.

Effect of Evodiamine on Cancer Metabolism of Liver Tumor Through Met/EGFR and HIF Pathways

Effect of Evodiamine on Cancer Metabolism of Liver Tumor Through Met/EGFR and HIF Pathways

A Network Pharmacology Perspective Investigation of the Pharmacological Mechanisms of the Herbal Drug FDY003 in Gastric Cancer

Gastric cancer (GC) is one of the most common and deadly malignant tumors worldwide. While the application of herbal drugs for GC treatment is increasing, the multicompound–multitarget pharmacological mechanisms involved are yet to be elucidated. By adopting a network pharmacology strategy, we investigated the properties of the anticancer herbal drug FDY003 against GC. We found that FDY003 reduced the viability of human GC cells and enhanced their chemosensitivity. We also identified 8 active phytochemical compounds in FDY003 that target 70 GC-associated genes and proteins. Gene ontology (GO) enrichment analysis suggested that the targets of FDY003 are involved in various cellular processes, such as cellular proliferation, survival, and death. We further identified various major FDY003 target GC-associated pathways, including PIK3-Akt, MAPK, Ras, HIF-1, ErbB, and p53 pathways. Taken together, the overall analysis presents insight at the systems level into the pharmacological activity of FDY003 against GC.

Co-Culturing of Osteoblasts and Chondrocytes Upregulates HIF-1 Pathway of Chondrocytes via MAPK Signaling

To study the effect of co-culturing chondrocytes with osteoblasts on hypoxia-inducible factor (HIF)-1 pathway of chondrocytes and its mechanism. Chondrocytes and osteoblasts were separately extracted from the knee joint and skull of newborn mice by trypsin digestion. The co-culturing system of osteoblasts and chondrocytes was constructed by using Transwell inserts to culture the osteoblasts and 6-well plate to culture the chondrocytes. We used qRT-PCR to examine changes in the mRNA expression of HIFs and its target gene pyruvate dehydrogenase kinase 1 ( PDK1) in chondrocytes co-cultured for 24 h. Western blot was used to analyze changes in the protein expression of HIFs and PDK1 and the changes in the activation of mitogen activated protein kinase (MAPK) signaling pathway after the cells were co-cultured for 48 h. Reactive oxygen species (ROS) staining was done to show the changes of ROS production in chondrocytes co-cultured for 48 h. The results of qRT-PCR and Western blot showed upregulated levels of HIF-1α gene and protein expression ( P<0.05) in the chondrocytes after they were co-cultured with osteoblasts. The gene and protein expression levels of PDK1 , the target gene of HIF-1, were also upregulated ( P<0.05). ROS staining showed that co-culturing of chondrocytes with osteoblasts decreased ROS production in chondrocytes. Western blot revealed that extracellular signal-regulated kinase (ERK) 1/2 and p38 signaling of co-cultured chondrocytes were enhanced ( P<0.05). Co-culturing with osteoblasts enhanced the ERK1/2 and p38 signaling of chondrocytes and upregulated the HIF-1 pathway of chondrocytes.

Interactions between phenolic constituents of Scutellaria salviifolia and key targets associated with inflammation: network pharmacology, molecular docking analysis and in vitro assays

Scutellaria salviifolia Benth. (SS), an endemic plant for Turkey, is used for gastric ailments as folk medicine. In this study, we aimed to uncover the underlying molecular mechanisms with the help of network pharmacology and molecular docking analysis in the inflammation processes of gastric ailments. Gene enrichment analysis and target screening were carried out. Experimental validation was performed via cytokines of nitric oxide (NO) and interleukin-6 (IL-6) in LPS stimulated RAW 264.7 cells. Furthermore, antioxidant activity studies were performed by radical scavenging effects on different radicals. A total of 144 targets were listed for the isolated compounds where 26 of them were related to selected inflammation targets. According to the gene enrichment analysis, HIF1 signaling pathway and TNF signaling pathway were found to be involved in inflammation. We also defined AKT1, TNF, EGFR, and COX2 as key targets due to the protein-protein interactions of 26 common targets. The extract inhibited NO and IL-6 production at 100 and 200 µg/mL, while flavonoid-rich fraction possessed significant anti-inflammatory activity at the concentration of 50 and 100 µg/mL via NO and IL-6 production, respectively. It is thought that the anti-inflammatory effects of extracts, fractions and pure compounds were achieved by reducing NO and IL-6 levels via regulating the NF-κB pathway or reducing NO production by suppressing iNOS through the HIF-1 pathway when evaluated together with the results of network analysis and literature. Anti-inflammatory activities of the extract and fractions were promising and comparably with S. baicalensis, commonly used for its anti-inflammatory activity.

Antiproliferative, Antiangiogenic, and Antimetastatic Therapy Response by Mangiferin in a Syngeneic Immunocompetent Colorectal Cancer Mouse Model Involves Changes in Mitochondrial Energy Metabolism.

In spite of the current advances and achievements in cancer treatments, colorectal cancer (CRC) persists as one of the most prevalent and deadly tumor types in both men and women worldwide. Drug resistance, adverse side effects and high rate of angiogenesis, metastasis and tumor relapse remain one of the greatest challenges in long-term management of CRC and urges need for new leads of anticancer drugs. We demonstrate that CRC treatment with the phytopharmaceutical mangiferin (MGF), a glucosylxanthone present in Mango tree stem bark and leaves (Mangifera Indica L.), induces dose-dependent tumor regression and decreases lung metastasis in a syngeneic immunocompetent allograft mouse model of murine CT26 colon carcinoma, which increases overall survival of mice. Antimetastatic and antiangiogenic MGF effects could be further validated in a wound healing in vitro model in human HT29 cells and in a matrigel plug implant mouse model. Interestingly, transcriptome pathway enrichment analysis demonstrates that MGF inhibits tumor growth, metastasis and angiogenesis by multi-targeting of mitochondrial oxidoreductase and fatty acid β-oxidation metabolism, PPAR, SIRT, NFκB, Stat3, HIF, Wnt and GP6 signaling pathways. MGF effects on fatty acid β-oxidation metabolism and carnitine palmitoyltransferase 1 (CPT1) protein expression could be further confirmed in vitro in human HT29 colon cells. In conclusion, antitumor, antiangiogenic and antimetastatic effects of MGF treatment hold promise to reduce adverse toxicity and to mitigate therapeutic outcome of colorectal cancer treatment by targeting mitochondrial energy metabolism in the tumor microenvironment.

Gene set enrichment analysis and ingenuity pathway analysis to identify biomarkers in Sheng-ji Hua-yu formula treated diabetic ulcers

Ethnopharmacological relevanceSheng-ji Hua-yu (SJHY) formula is a Chinese herbal prescription for diabetic ulcers (DUs) treatment, which can accelerate wound reconstruction and shorten the healing time. However, its mechanism role maintains unclear. Aim of the studyTo elucidate the molecular mechanisms of SJHY application on DUs. Materials and methodsTo begin with, transcriptome sequencing was adopted to identified differentially expression mRNAs among normal ulcers, DUs, and DUs + SJHY treatment in vivo. Liquid chromatography-tandem mass spectrometry was applied for the quality control of SJHY formula. GO and KEGG enrichment analysis were used to identify the mechanisms underlying the therapeutic effect of SJHY formula, and then gene set enrichment analysis and ingenuity pathway analysis were conducted for functional analysis. Further, qPCR detection was performed in vivo for validation. ResultsSJHY administration could regulate the glucose metabolic process, AMPK and HIF-1 pathway to accelerate healing processes of DUs. Besides, CRHR1, SHH, and GAL were identified as the critical targets, and SLC6A3, GRP, FGF23, and CYP27B1 were considered as the upstream genes of SJHY treatment. Combined with animal experiments, the prediction results were validated in DUs mice model. ConclusionsThis study used modular pharmacology analysis to identify the biomarkers of SJHY formula and provide the potential therapeutic targets for DUs treatment as well.

Treatment with sodium (S)-2-hydroxyglutarate prevents liver injury in an ischemia-reperfusion model in female Wistar rats

BackgroundIschemia-reperfusion (IR) injury is one of the leading causes of early graft dysfunction in liver transplantation. Techniques such as ischemic preconditioning protect the graft through the activation of the hypoxia-inducible factors (HIF), which are downregulated by the EGLN family of prolyl-4-hydroxylases, a potential biological target for the development of strategies based on pharmacological preconditioning. For that reason, this study aims to evaluate the effect of the EGLN inhibitor sodium (S)-2-hydroxyglutarate [(S)-2HG] on liver IR injury in Wistar rats.MethodsTwenty-eight female Wistar rats were divided into the following groups: sham (SH, n = 7), non-toxicity (HGTox, n = 7, 25 mg/kg of (S)-2HG, twice per day for two days), IR (n = 7, total liver ischemia: 20 minutes, reperfusion: 60 minutes), and (S)-2HG+IR (HGIR, n = 7, 25 mg/kg of (S)-2HG, twice per day for two days, total liver ischemia as the IR group). Serum ALT, AST, LDH, ALP, glucose, and total bilirubin were assessed. The concentrations of IL-1β, IL-6, TNF, malondialdehyde, superoxide dismutase, and glutathione peroxidase were measured in liver tissue, as well as the expression of Hmox1, Vegfa, and Pdk1, determined by RT-qPCR. Sections of liver tissue were evaluated histologically, assessing the severity of necrosis, sinusoidal congestion, and cytoplasmatic vacuolization.ResultsThe administration of (S)-2HG did not cause any alteration in the assessed biochemical markers compared to SH. Preconditioning with (S)-2HG significantly ameliorated IR injury in the HGIR group, decreasing the serum activities of ALT, AST, and LDH, and the tissue concentrations of IL-1β and IL-6 compared to the IR group. IR injury decreased serum glucose compared to SH. There were no differences in the other biomarkers assessed. The treatment with (S)-2HG tended to decrease the severity of hepatocyte necrosis and sinusoidal congestion compared to the IR group. The administration of (S)-2HG did not affect the expression of Hmox1 but decreased the expression of both Vegfa and Pdk1 compared to the SH group, suggesting that the HIF-1 pathway is not involved in its mechanism of hepatoprotection. In conclusion, (S)-2HG showed a hepatoprotective effect, decreasing the levels of liver injury and inflammation biomarkers, without evidence of the involvement of the HIF-1 pathway. No hepatotoxic effect was observed at the tested dose.

TAMI-43. HYPOXIC REGULATION OF GLIOBLASTOMA TUMOR GROWTH THROUGH L(3)MBTL1

Abstract Glioblastoma (GBM) is the deadliest and most common of all primary brain tumors. Drosophila brain tumor models have uncovered signaling pathways regulating tumor growth that are highly conserved in GBM. Our search for a novel tumor suppressor using Drosophila led to Lethal (3) malignant brain tumor [l(3)mbt], temperature-sensitive mutants of which cause neuroepithelial tumor-like overproliferation in optic lobes. dL(3)MBT and its human orthologs L3MBTL1-L3MBTL4 all harbor Malignant Brain Tumor (MBT) domains that recognize methylated lysines on histone tails. Like dL(3)MBT, hL(3)MBTL1 acts as a chromatin compaction factor that represses transcription and inhibits cytokinesis in GBM cell lines. The highly hypoxic tumor microenvironment (TME) in GBM drives its progression, recurrence, and therapeutic resistance. However, it remains unclear if L(3)MBTL1 is regulated by TME cues to promote GBM growth. Based on this knowledge gap and our preliminary data, we hypothesize that hypoxia directly regulates L(3)MBTL1 in favor of GBM growth. Analysis of TCGA data for IDH-wildtype gliomas revealed that L3MBTL1 gene expression is downregulated in GBM, which are necrotic and severely hypoxic, compared to histologic grade 2/3 gliomas, which do not contain necrosis, indicating that hypoxia could potentially suppress L3MBTL1 to enhance glioma progression. TCGA data also revealed a number of HIF pathway and hypoxia-inducible genes strongly correlating with L3MBTL1 expression, including HIF1a and VHL. Using patient-derived GBM neurosphere cultures, we exposed glioma cells to hypoxia (1% O2 for 24hrs) and found that L3MBTL1 protein levels were suppressed compared to normoxia (21%). Under these same conditions, we found more rapid cell proliferation under hypoxia. Exploration of hypoxic TME regulation of the novel tumor suppressor L3MBTL1 in glioma progression has the potential to uncover novel mechanisms involving epigenetic modulation and potentially new therapeutic strategies.

Efficacy of Allium Sativum, Curcuma Longa and Zingiber Officinale Extract on Serum Vasculoendothelial Growth Factors Levels of Breast Cancer Patients

ntroduction: Breast cancer requires high nutrition, oxygen and metabolism where VEGF-triggered angiogenesis is needed. VEGF is triggered via the HIF-1 pathway and also via the NF-Kappa B pathway. VEGF is associated with tumor growth and metastasis. Some plant extracts have anti-angiogenesis effects such as Curcuma longa, Allium sativum and Zingiber officinale. Objective: To determine serum levels of SGOT, SGPT, ureum, creatinine, albumin and VEGF before and after administration of Curcuma longa, Allium sativum and Zingiber officinale extracts Method: An open label clinical trial study with a one group pretest posttest design involving 21 patients from breast cancer community in Medan collected by consecutive sampling from June – July 2021 in USU hospital where these patients met the inclusion criteria. All patients were tested for complete blood count, urea, creatinine, SGOT, SGPT, albumin, serum VEGF before and after being given a combination extract of Curcuma longa 150 mg, Allium sativum 200 mg and Zingiber officinale 150 mg twice daily for 28 days. Statistical analysis was performed by paired analytical T test or Wilcoxon. Results: The study involved 20 subjects. From the examination of kidney function obtained results before and after the administration of a combination extract VEGF results before and after the administration of the combination (1110(760-7893) μg / ml vs. 864 (532-5650 μg / ml, p = 0.001). Conclusion: VEGF serum breast cancer patients had a significant reduction after being given a combination of extracts of Allium sativum, Curcuma longa and Zingber officinale. Keywords: VEGF, Allium sativum, Curcuma longa, Zingiber officinale.