Shikonin induces hepatocellular carcinoma cell apoptosis by suppressing PKM2/PHD3/HIF-1α signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism of shikonin-induced death of human hepatocellular carcinoma SMMC-7721 cells.@*METHODS@#Cultured SMMC-7721 cells and normal hepatocytes (L-02 cells) were treated with 4, 8, or 16 μmol/L shikonin, and the changes in cell viability was assessed using MTT assay. The levels of ATP and lactic acid in the cell cultures were detected using commercial kits. Co-immunoprecipitation and immunofluorescence staining were used to determine the relationship among pyruvate kinase M2 (PKM2), prolyl hydroxylase 3 (PHD3), and hypoxia-inducible factor-1α (HIF-1α). The expressions of PHD3, PKM2, HIF-1α, Bax, cleaved caspase-3, and Bcl-2 in SMMC-7721 cells were detected with Western blotting, and cell apoptosis was analyzed with annexin V-FITC/PI staining. The effects of RNA interference of PKM2 on PHD3 and HIF-1α expressions in SMMC-7721 cells were detected using Western blotting.@*RESULTS@#The IC50 of shikonin against SMMC-7721 and L-02 cells was 8.041 μmol/L and 31.75 μmol/L, respectively. Treatment with shikonin significantly inhibited the protein expressions of PKM2, HIF-1α and PHD3 and nuclear translocation of PKM2 and HIF-1α in SMMC-7721 cells. Coimmunoprecipitation and immunofluorescence staining confirmed that shikonin inhibited the formation of PKM2/PHD3/HIF-1α complex and significantly reduced the contents of lactic acid and ATP in SMMC-7721 cells (P < 0.05). The expressions of PHD3 and HIF-1α decreased significantly after PKM2 knockdown (P < 0.05). Shikonin treatment significantly increased the apoptosis rate, enhanced the expressions of Bax and cleaved caspase-3, and decreased Bcl-2 expression in SMMC-7721 cells (P < 0.05).@*CONCLUSIONS@#Shikonin induces apoptosis of SMMC-7721 cells possibly by inhibiting aerobic glycolysis through the PKM2/PHD3/HIF-1α signaling pathway to cause energy supply dysfunction in the cells.

CHD1 deletion stabilizes HIF1α to promote angiogenesis and glycolysis in prostate cancer / 亚洲男科学杂志(英文版)

Chromodomain-helicase-DNA-binding protein 1 (CHD1) deletion is among the most common mutations in prostate cancer (PCa), but its role remains unclear. In this study, RNA sequencing was conducted in PCa cells after clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-based CHD1 knockout. Gene set enrichment analysis (GSEA) indicated upregulation of hypoxia-related pathways. A subsequent study confirmed that CHD1 deletion significantly upregulated hypoxia-inducible factor 1α (HIF1α) expression. Mechanistic investigation revealed that CHD1 deletion upregulated HIF1α by transcriptionally downregulating prolyl hydroxylase domain protein 2 (PHD2), a prolyl hydroxylase catalyzing the hydroxylation of HIF1α and thus promoting its degradation by the E3 ligase von Hippel-Lindau tumor suppressor (VHL). Functional analysis showed that CHD1 deletion promoted angiogenesis and glycolysis, possibly through HIF1α target genes. Taken together, these findings indicate that CHD1 deletion enhances HIF1α expression through PHD2 downregulation and therefore promotes angiogenesis and metabolic reprogramming in PCa.

P4HA2 promotes occurrence and progression of liver cancer by regulating the PI3K/Akt/mTOR signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the role of proline 4-hydroxylase Ⅱ (P4HA2) in the occurrence and progression of liver cancer.@*METHODS@#GEPIA and Human Protein Atlas database were used to predict the expression of P4HA2 in hepatocellular carcinoma (HCC), and K-M plotter online database was used to analyze the relationship between P4HA2 expression and the prognosis of HCC. We also examined the expressions of P4HA2 in HCC cells and normal hepatocytes using qRT-PCR and Western blotting. With lentivirus-mediated RNA interference, P4HA2 expression was knocked down in hepatoma SNU-449 and Hep-3B cells, and the changes in cell proliferation, migration and invasion were assessed using cell counting kit-8 (CCK-8) assay, colony formation test, scratch test and Transwell assay. The changes in the expressions of epithelial-mesenchymal transition (EMT) and PI3K/Akt/mTOR signal pathway-related proteins were detected using Western blotting.@*RESULTS@#Online database analysis showed that the expression of P4HA2 was significantly higher in HCC tissues than in normal liver tissues (P < 0.05). The expression levels of P4HA2 mRNA and protein were also significantly higher in HCC cell lines than in normal hepatocytes (P < 0.01). Lentivirus-mediated RNA interference of P4HA2 significantly lowered the expression levels of P4HA2 mRNA and protein in the hepatoma cells (P < 0.05) and caused obvious inhibition of cell proliferation, migration and invasion. P4HA2 knockdown significantly increased the expression of E-cadherin protein, lowered the expressions of N-cadherin and Snail, and obviously decreased the expressions of phosphorylated PI3K, AKT and mTOR (P < 0.05).@*CONCLUSION@#P4HA2 enhances the proliferation, migration, invasion, and EMT of hepatoma cells by activating the PI3K/Akt/mTOR signaling pathway to promote the occurrence and progression of liver cancer.

Research progress on the role of HIFs-PHDs oxygen-sensing pathway in cellular ferroptosis / 生理学报

Hypoxia-inducible factors (HIFs) are one of the primary transcription factors regulating oxygen balance, and their stability is determined by the hydroxylation state of the prolyl hydroxylase domain (PHD) that is sensitive to oxygen. In recent years, studies have shown that HIFs-prolyl hydroxylases (PHDs) oxygen-sensing pathway is involved in the process of cellular ferroptosis. Ferroptosis, a new type of cell death, different from necrosis, apoptosis, necrotizing apoptosis, and pyroptosis, is essentially a programmed death caused by the accumulation of iron-dependent lipid peroxides in cells. This paper focuses on the role and mechanism of the HIFs-PHDs oxygen-sensing pathway in cellular ferroptosis involved in nerve diseases, tumors, lung injury, and chemical nerve damage from three aspects of iron metabolism, lipid metabolism, and glutathione (GSH) synthesis/metabolism. This review will provide a theoretical basis and new ideas for the development of novel drugs targeting the HIFs-PHDs oxygen-sensing pathway and capable of regulating ferroptosis for the treatment of diseases related to ferroptosis such as nervous system diseases and tumors.

New Oral Agent for Treatment of Anemia in Patient with Chronic Kidney Disease: Prolyl Hydroxylase Inhibitor / 대한내과학회지

Hypoxia inducible factor (HIF)-stabilizers are being developed for the renal anemia treatment. This small molecules inhibit prolyl hydroxylase domain (PHD)-containing enzymes, causing HIF activation instead of degradation under the state of normoxia, finally increase production of intrinsic erythropoiesis. Current treatment guidelines suggest that renal anemia should be treated mainly with iron and erythropoiesis stimulating agents (ESAs). But there are several complications and concerns such as hypertension, ESA refractory anemia and increased cardiovascular mortality in using ESAs. Advantages of HIF stabilizers over ESAs are orally available, no dose-up requirement for inflammation. So far new HIF stabilizers showed efficacy and safety in renal anemia treatment. This new therapeutic agent may emerge as a standard treatment option for renal anmia treatment.

Current insights into the role of HIF-PHD axis in renal anemia / 生理学报

Renal anemia, mainly caused by the deficiencies of erythropoietin (EPO) and iron metabolism disorder, is one of the most common complications of chronic kidney disease. Hypoxia-inducible factor (HIF) is a class of transcription factors responsible for maintaining homeostasis during oxygen deprivation. In normoxia, HIF is degraded by prolyl hydroxylase (PHD). While under hypoxic conditions, the hydroxylation activity of PHD is inhibited, and the cellular concentration of HIF is elevated, resulting in an increase in endogenous EPO production and iron absorption. Therefore, this regulating pathway, also termed as the HIF-PHD axis, has become a promising therapeutic target of treating renal anemia. Several innovative drugs acting as selective HIF-PHD inhibitors have been successfully developed in the past years, and some of them are undergoing clinical trials. In this review, we will introduce the definition and regulatory mechanism of HIF-PHD axis, as well as current insights into its physiologic and therapeutic role in renal anemia.

Expression of prolyl 4-hydroxylase beta-polypeptide in non-small cell lung cancer treated with Chinese medicines / 中国结合医学杂志

<p><b>OBJECTIVES</b>To investigate the role of prolyl 4-hydroxylase beta polypeptide (P4HB) expressed in lung carcinoma and the intervention effect of Yiqi Chutan Formula (, YQCTF).</p><p><b>METHODS</b>Lung carcinoma model was established by subcutaneously inoculating LEWIS lung carcinoma cells in C57BL/6J mice. The differential expression of P4HB protein between the YQCTF (3.0 g/kg, gavage, once daily, 21 days) group and the control group was acquired by a 2 fluorescence difference gel electrophoresis (2D-DIGE), verified by Western blotting and identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS). The expression of P4HB and P4HB mRNA in cultured A549 cells from cisplatin (DDP) 1.5 µg/mL group and 15% serum combined with DDP 1.5 µg/mL group were detected by cellular immunohistochemistry and reverse transcription-polymerase chain reaction, respectively.</p><p><b>RESULTS</b>The proteomics research discovered that one-third of differential proteins including P4HB were decreased in the YQCTF group (P<0.01). Clinical pathology and tissue microarray studies showed that P4HB expression in lung cancer tissue was stronger than adjacent tissues and normal lung epithelial (P<0.01). In the YQCTF and DDP combined groups, the expression of P4HB and P4HB mRNA in A549 cell were decreased significantly (P<0.01).</p><p><b>CONCLUSION</b>YQCTF could inhibit the LEWIS lung carcinoma's growth, decrease the expression of P4HB in LEWIS lung carcinoma and A549 cells. YQCTF might take effect through regulating P4HB in endoplasmic reticulum to inhibit the incidence and growth process of lung carcinoma.</p>

Profiling of Proteins Regulated by Venlafaxine during Neural Differentiation of Human Cells

OBJECTIVE: Antidepressants are known to positively influence several factors in patients with depressive disorders, resulting in increased neurogenesis and subsequent relief of depressive disorders. To study the effects of venlafaxine during neural differentiation at the cellular level, we looked at its effect on protein expression and regulation mechanisms during neural differentiation. METHODS: After exposing NCCIT cell-derived EBs to venlafaxine during differentiation (1 day and 7 days), changes in protein expression were analyzed by 2-DE and MALDI-TOF MS analysis. Gene levels of proteins regulated by venlafaxine were analyzed by real-time RT-PCR. RESULTS: Treatment with venlafaxine decreased expression of prolyl 4-hydroxylase (P4HB), ubiquitin-conjugating enzyme E2K (HIP2) and plastin 3 (T-plastin), and up-regulated expression of growth factor beta-3 (TGF-beta3), dihydropyrimidinase-like 3 (DPYSL3), and pyruvate kinase (PKM) after differentiation for 1 and 7 days. In cells exposed to venlafaxine, the mRNA expression patterns of HIP2 and PKM, which function as negative and positive regulators of differentiation and neuronal survival, respectively, were consistent with the observed changes in protein expression. CONCLUSION: Our findings may contribute to improve understanding of molecular mechanism of venlafaxine.

Phospholipase D2 promotes degradation of hypoxia-inducible factor-1alpha independent of lipase activity

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key transcriptional mediator that coordinates the expression of various genes involved in tumorigenesis in response to changes in oxygen tension. The stability of HIF-1alpha protein is determined by oxygen-dependent prolyl hydroxylation, which is required for binding of the von Hippel-Lindau protein (VHL), the recognition component of an E3 ubiquitin ligase that targets HIF-1alpha for ubiquitination and degradation. Here, we demonstrate that PLD2 protein itself interacts with HIF-1alpha, prolyl hydroxylase (PHD) and VHL to promote degradation of HIF-1alpha via the proteasomal pathway independent of lipase activity. PLD2 increases PHD2-mediated hydroxylation of HIF-1alpha by increasing the interaction of HIF-1alpha with PHD2. Moreover, PLD2 promotes VHL-dependent HIF-1alpha degradation by accelerating the association between VHL and HIF-1alpha. The interaction of the pleckstrin homology domain of PLD2 with HIF-1alpha also promoted degradation of HIF-1alpha and decreased expression of its target genes. These results indicate that PLD2 negatively regulates the stability of HIF-1alpha through the dynamic assembly of HIF-1alpha, PHD2 and VHL.