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Background Acute myocardial infarction (AMI) is a leading cause of mortality, characterized by myocardial ischemia that induces cardiomyocyte apoptosis and subsequent cardiac dysfunction. Sirtuin 1 (Sirt1) has emerged as a key regulator of cell survival and apoptosis, particularly under hypoxic conditions. Methods An AMI animal model was established via ligation of the left anterior descending (LAD) coronary artery. Gene expression in the infarcted region was evaluated at various time points. Sirt1 overexpression and control lentivirus were administered to the peri-infarct region of mice heart. After LAD ligation, assessment on myocardial infarct size, cardiac function, and cardiomyocyte apoptosis were performed. In vitro, primary mouse cardiomyocytes subjected to hypoxia were analyzed for gene expression, while interactions among Sirt1, Phd3, and Hif-1α were explored using diverse treatment approaches. Results A significant reduction in Sirt1 and Phd3 expression, along with an increase in Hif-1α and cleaved caspase-3, was observed in a time-dependent manner post-myocardial infarction (MI). In vitro findings revealed that hypoxia decreased nuclear Sirt1 and cytoplasmic Phd3 levels while promoting a time-dependent increase in Hif-1α and cleaved caspase-3. Furthermore, Sirt1 overexpression enhanced Phd3 expression in cardiomyocytes, suppressed Hif-1α and cleaved caspase-3 levels, and alleviated hypoxia-induced cardiomyocyte apoptosis. Notably, knockdown of Phd3 negated Sirt1’s inhibitory effect on Hif-1α, whereas Hif-1α knockdown promoted Sirt1 expression. Sirt1 overexpression reduced infarct size, decreased cardiomyocyte apoptosis, and improved cardiac function. Conclusions Sirt1 effectively reduces cardiomyocyte apoptosis and myocardial infarction size while enhancing cardiac function post-MI, primarily through the Phd3/Hif-1α signaling pathway.

Metformin has been under basic and clinical study as an oncological repurposing pharmacological agent for several years, stemming from observational studies which consistently evidenced that subjects who were treated with metformin had a reduced risk for development of cancer throughout their lives, as well as improved survival outcomes when diagnosed with neoplastic diseases. As a result, several basic science studies have attempted to dissect the relationship between metformin’s metabolic mechanism of action and antineoplastic cellular signaling pathways. Evidence in this regard was compelling enough that a myriad of randomized clinical trials was planned and conducted in order to establish the effect of metformin treatment for patients with diverse neoplasms, including lung cancer. As with most novel antineoplastic agents, early results from these studies have been mostly discouraging, though a recent analysis that incorporated body mass index may provide significant information regarding which patient subgroups might derive the most benefit from the addition of metformin to their anticancer treatment. Much in line with the current pipeline for anticancer agents, it appears that the benefit of metformin may be circumscribed to a specific patient subgroup. If so, addition of metformin to antineoplastic agents could prove one of the most cost-effective interventions proposed in the context of precision oncology. Currently published reviews mostly rely on a widely questioned mechanism of action by metformin, which fails to consider the differential effects of the drug in lean vs. obese subjects. In this review, we analyze the pre-clinical and clinical information available to date regarding the use of metformin in various subtypes of lung cancer and, further, we present evidence as to the differential metabolic effects of metformin in lean and obese subjects where, paradoxically, the obese subjects have reported more benefit with the addition of metformin treatment. The novel mechanisms of action described for this biguanide may explain the different results observed in clinical trials published in the last decade. Lastly, we present novel hypothesis regarding potential biomarkers to identify who might reap benefit from this intervention, including the role of prolyl hydroxylase domain 3 (PHD3) expression to modify metabolic phenotypes in malignant diseases.

The prolyl hydroxylase 3 (PHD3) protein is less abundant in normal oxygen conditions (normoxia) but increases under deficient oxygen condition (hypoxia). Since cancerous cells often thrive in hypoxic conditions and predominantly express the Pyruvate kinase isoforms 2 (PKM2), the PHD3/PKM2 interaction might be particularly important in cancer development. In the present study, the PHD3/PKM2 complex was co-expressed and purified by size-exclusion chromatography. The interaction of PHD3 with PKM2 was confirmed in Native gel as well as western blot analysis. The PHD3/PKM2 complex formed discreet crystals under suitable conditions, and diffraction data revealed that crystal belonged to the P1 space group with 3.0 Å resolution. This is the first crystal report of PHD3/PKM2 complex as well as this study demonstrates a direct physical binding through protein–protein interaction. The structural analysis of complex will provide the information regarding the amino acid residues critical for the catalytic mechanism. Based on the structural information thus obtained, pharmacological interference with the PHD3/PKM2 interaction could be used as a novel strategy to reduce the cancer progression.

The mitogen-activated protein kinase organizer 1 (MORG1) is a scaffold molecule for the ERK signaling pathway, but also binds to prolyl-hydroxylase 3 and modulates HIFα expression. To obtain further insight into the role of MORG1, knockout-mice were generated by homologous recombination. While Morg1+/− mice developed normally without any apparent phenotype, there were no live-born Morg1−/− knockout offspring, indicating embryonic lethality. The intrauterine death of Morg1−/− embryos is caused by a severe failure to develop brain and other neuronal structures such as the spinal cord and a failure of chorioallantoic fusion. On E8.5, Morg1−/− embryos showed severe underdevelopment and proliferative arrest as indicated by absence of Ki67 expression, impaired placental vascularization and altered phenotype of trophoblast giant cells. On E9.5, the malformed Morg1−/− embryos showed defective turning into the final fetal position and widespread apoptosis in many structures. In the subsequent days, apoptosis and decomposition of embryonic tissue progressed, accompanied by a massive infiltration of inflammatory cells. Developmental aberrancies were accompanied by altered expression of HIF-1/2α and VEGF-A and caspase-3 activation in embryos and extraembryonic tissues. In conclusion, the results suggest a multifactorial process that causes embryonic death in homozygous Morg1 mutant mice, described here, to the best of our knowledge, for the first time.

Background Solid tumours are less oxygenated than normal tissues. Consequently, cancer cells acquire to be adapted to a hypoxic environment. The poor oxygenation of solid tumours is also a major indicator of an adverse cancer prognosis and leads to resistance to conventional anticancer treatments. We previously showed the involvement of Che-1/AATF (Che-1) in cancer cell survival under stress conditions. Herein we hypothesized that Che-1 plays a role in the response of cancer cells to hypoxia. Methods The human colon adenocarcinoma HCT116 and HT29 cell lines undepleted or depleted for Che-1 expression by siRNA, were treated under normoxic and hypoxic conditions to perform studies regarding the role of this protein in metabolic adaptation and cell proliferation. Che-1 expression was detected using western blot assays; cell metabolism was assessed by NMR spectroscopy and functional assays. Additional molecular studies were performed by RNA seq, qRT-PCR and ChIP analyses. Results Here we report that Che-1 expression is required for the adaptation of cells to hypoxia, playing an important role in metabolic modulation. Indeed, Che-1 depletion impacted on HIF-1α stabilization, thus downregulating the expression of several genes involved in the response to hypoxia and affecting glucose metabolism. Conclusions We show that Che-1 a novel player in the regulation of HIF-1α in response to hypoxia. Notably, we found that Che-1 is required for SIAH-2 expression, a member of E3 ubiquitin ligase family that is involved in the degradation of the hydroxylase PHD3, the master regulator of HIF-1α stability.

Purpose: Tumour hypoxia activates hypoxia-inducible factor-1 (HIF-1) and indluences angiogenesis, cell survival and invasion. Prolyl hydroxylase-3 (PHD3) regulates degradation of HIF-1 α . The effects of PHD3 in tumour growth are largely unknown. Experimental design: PHD3 expression was analysed in human pancreatic cancer tissues and cancer cell lines by real-time quantitative PCR and immunohistochemistry. PHD3 overexpression was established by stable transfection and downregulation by short interfering RNA technology. VEGF was quantified by enzyme-linked immunosorbent assay. Matrigel invasion assays were performed to examine tumour cell invasion. Apoptosis was measured by annexin-V staining and caspase-3 assays. The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model. Results: PHD3 was upregulated in well-differentiated human tumours and cell lines, and regulated hypoxic VEGF secretion. PHD3 overexpression mediated tumour cell growth and invasion by induction of apoptosis in a nerve growth factor-dependent manner by the activation of caspase-3 and phosphorylation of focal adhesion kinase HIF-1 independently. In vivo , PHD3 inhibited tumour growth by abrogation of tumour angiogenesis. Conclusion: Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.

It has been reported that by regulating PHD3 stability, Siah1 contributes to the abundance of hypoxia-inducible factor (HIF)-1α, thereby playing an important role in the cellular response to hypoxia. However, the expression level and functional significance of Siah1 in human malignant glioma, which is characterized by high migration and invasion potential, have never been investigated. We report here, that Siah1 was expressed highly in human glioma tissues compared with its expression in normal brain tissues and was correlated with advanced tumor status and stage. The knockdown of Siah1 by short-hairpin RNA severely suppressed the migration and invasion of human glioma U251 cells under hypoxia, while overexpression of Siah1 promoted it. Furthermore, we demonstrated that the glioma cell migration and invasion under hypoxia mediated by Siah1 was achieved by reducing the stability of PHD3, which protected the HIF-1α from degradation. These findings suggest that Siah1 plays important roles in the migration and invasion of human glioma cells under hypoxia, which may provide some guidance for the targeted therapy of human glioma based on the interference of the Siah1-PHD3-HIF-1α signaling pathway.

Pancreatic cancer is characterized by a hypoxic microenvironment and resistance to most currently available treatment modalities. Prolyl hydroxylase domain 3 (PHD3) is a rate-limiting enzyme that regulates the degradation of hypoxia-inducible factors (HIFs) and is deregulated in pancreatic cancer cells. Whether such alteration of PHD3 expression contributes to the sustained growth and radioresistance of pancreatic cancer cells remains largely unknown. PHD3 was overexpressed in pancreatic cancer Mia-paca2 cells via lentiviral expression. Cell cycle progression and apoptosis were assayed by flow cytometry. HIF-1α, EGFR, and PHD3 protein expression was assessed by Western blotting. Cell survival was determined in a colony formation assay. PHD3 overexpression suppressed HIF-1α protein expression and EGFR phosphorylation and enhanced the 2 Gy irradiation-mediated reductions in HIF-1α and phosphorylated (p)-EGFR under either normoxic or hypoxic conditions. PHD3 overexpression inhibited the growth and colony formation of Mia-paca2 cells in response to irradiation under either normoxic or hypoxic conditions. PHD3 overexpression exacerbated irradiation-induced apoptosis, with a greater effect under hypoxia than normoxia. Cell cycle distribution analysis demonstrated that PHD3 overexpression resulted in further shortened S phase and lengthened G2/M phase in response to irradiation. PHD3 expression may contribute to the radiotherapy efficacy of pancreatic cancer cells and serve as a novel biomarker for improving radiotherapy efficacy in pancreatic cancer.

Background A key feature of clear cell renal cell carcinoma (ccRCC) is the inactivation of the von Hippel-Lindau tumour suppressor protein (pVHL) that leads to the activation of hypoxia-inducible factor (HIF) pathway also in well-oxygenated conditions. Important regulator of HIF-α, prolyl hydroxylase PHD3, is expressed in high amounts in ccRCC. Although several functions and downstream targets for PHD3 in cancer have been suggested, the role of elevated PHD3 expression in ccRCC is not clear. Methods To gain insight into the functions of high PHD3 expression in ccRCC, we used PHD3 knockdown by siRNA in 786-O cells under normoxic and hypoxic conditions and performed discovery mass spectrometry (LC-MS/MS) of the purified peptide samples. The LC-MS/MS results were analysed by label-free quantification of proteome data using a peptide-level expression-change averaging procedure and subsequent gene ontology enrichment analysis. Results Our data reveals an intriguingly widespread effect of PHD3 knockdown with 91 significantly regulated proteins. Under hypoxia, the response to PHD3 silencing was wider than under normoxia illustrated by both the number of regulated proteins and by the range of protein expression levels. The main cellular functions regulated by PHD3 expression were glucose metabolism, protein translation and messenger RNA (mRNA) processing. PHD3 silencing led to downregulation of most glycolytic enzymes from glucose transport to lactate production supported by the reduction in extracellular acidification and lactate production and increase in cellular oxygen consumption rate. Moreover, upregulation of mRNA processing-related proteins and alteration in a number of ribosomal proteins was seen as a response to PHD3 silencing. Further studies on upstream effectors of the translational machinery revealed a possible role for PHD3 in regulation of mTOR pathway signalling. Conclusions Our findings suggest crucial involvement of PHD3 in the maintenance of key cellular functions including glycolysis and protein synthesis in ccRCC.

Introduction: Hypoxia inducible factors (HIF) and prolyl hydroxylase domain (PHD) enzymes play a central role in tumor progression in clear cell renal cell carcinoma (ccRCC). However, there are currently no data regarding the behavior of this pathway (HIF/PHD) in a large number of benign renal tumors, the oncocytomas. The aim of the present study was to compare the expression levels of these factors between ccRCC and oncocytoma tumors. Material and methods: A total of 56 fresh frozen specimens from patients with ccRCC and 14 oncocytoma specimens were analyzed via reverse transcription-quantitative polymerase chain reaction in order to assess the expression levels of HIF-1α, HIF-2α, PHD1, PHD2, and PHD3. The analysis involved both fresh frozen tumor samples as well as adjacent normal kidney tissues. Results: In ccRCC, HIF-1α and HIF-2α levels were upregulated in 65.5% and 71.4% of cases, respectively. PHD3 was downregulated only in 15.4% of the ccRCC cases, in contrast with oncocytoma cases, which exhibited low expression levels in the majority. The upregulation of PHD3 messenger RNA (mRNA) levels in ccRCC when compared with oncocytoma was statistically significant (P<0.001). No other comparisons (HIF-1α, HIF-2α, PHD1, and PHD2) were significantly different. HIF-2α and PHD3 mRNA expression levels were negatively correlated with Fuhrman Grade (P=0.029 and P=0.026, respectively) in ccRCC. Conclusion: To the best of our knowledge, this is the first time that the HIF/PHD pathway was compared between ccRCC and a common benign tumor, identifying the upregulation of PHD3 as the possible underlying factor guiding the difference in the behavior of ccRCC.