Hypoxia-inducible Factor Signaling Pathway Research Articles

HIF Inhibitors: Status of Current Clinical Development.

In this review, the importance of the hypoxia inducible factor (HIF) pathway in tumorigenesis and cancer treatment outcomes will be discussed. The outcomes of phase II and III clinical trials of direct HIF inhibitors in the treatment of cancer will be reviewed. The HIF signaling pathway is activated by tumor-induced hypoxia or by inactivating mutations of the VHL gene. HIF is a transcription factor which regulates the expression of genes involved in adjusting mechanisms to hypoxia such as angiogenesis or apoptosis as well as tumor growth, invasion, and metastasis. The HIF pathway has a key role in development of resistance to different treatment modalities and higher expression of the HIF molecule is associated with poor prognosis. Clinical studies of the HIF inhibitors in patients with advanced/refractory cancers suggest benefit and warrant further studies of the HIF inhibitors either as a single agent or in combination with other therapeutic agents.

Yak-milk-derived exosomes promote proliferation of intestinal epithelial cells in an hypoxic environment

Intestinal epithelial cells (IEC) are an important part of the intestinal barrier. Barrier function was disrupted under hypoxia, but milk-derived exosomes can regulate the intestinal barrier function. However, the mechanisms underlying the association between yak milk exosomes and hypoxia in IEC remain poorly understood. In this follow-up study, we proposed an effective optimization method for purifying yak-milk-derived exosomes. The Western blot analyses indicated that the expression of the proteins of the endosomal sorting complexes required for transport (TSG101), proteins of the tetraspanin family (CD63), and heat shock protein 70 (Hsp-70) proteins from yak-milk-derived exosomes were significantly higher than those in cow-milk-derived exosomes. Flow cytometry analysis showed that yak milk had 3.7 times the number of exosomes compared with cow milk. Moreover, we explored whether yak milk exosomes could facilitate intestinal cell survival under hypoxic conditions in vitro. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide results showed that yak-milk-derived exosomes significantly increased survival of IEC-6 cells with rates of up to 29% for cells incubated in hypoxic conditions for 12 h, compared with those of cow-milk-derived exosomes posttreatment (rates of up to 22% for cells incubated in hypoxic conditions for 12 h). Confocal microscopy revealed that the IEC-6 cells uptake more yak-milk-derived exosomes than cow milk in hypoxic conditions. Furthermore, the Western blot analyses indicated that yak-milk-derived exosomes significantly promote oxygen-sensitive prolyl hydroxylase (PHD)-1 expression and decrease the expression of hypoxia-inducible factor-α and its downstream target vascular endothelial growth factor (VEGF) in the IEC-6 cells. Further, yak-milk-derived exosomes significantly inhibited p53 levels. In conclusion, our findings demonstrate that yak-milk-derived exosomes more effectively activate the hypoxia-inducible factor signaling pathway, thus promoting IEC-6 cell survival, which may result in higher hypoxia tolerance than cow-milk-derived exosomes.

Long non coding RNAs in regulation of hypoxic program in heart

Background: Significant role in the pathogenesis of hypoxic status is given to the hypoxia-inducible factor (HIF). One of the likely mechanisms for regulating the HIF signaling pathway is long non-coding RNAs (lncRNAs). These molecules represent a significant part of the transcript and perform numerous regulatory functions at transcriptional and posttranscriptional levels.

Hypoxia-inducible Factor may Induce the Development of Liver Fibrosis in Budd-Chiari Syndrome by Regulating CD248/endosialin Expression: A Hypothesis.

Budd-Chiari syndrome (BCS) leads to the development of liver fibrosis in most of the cases. However, the mechanism of BCS-related liver fibrosis is unclear, and it may be largely different from that induced by chronic viral hepatitis. Hepatic stellate cell (HSC) and its specific marker CD248/endosialin are known to play an important regulatory role in the development of liver fibrosis. Additionally, hypoxia microenvironment and hypoxia-inducible factor (HIF) are involved in the regulation of CD248/endosialin. Therefore, we hypothesize that hypoxia microenvironment which develops due to BCS can regulate the expression of CD248/endosialin in HSC via HIF signaling pathway, which then affects the function of HSC and development of liver fibrosis. To confirm the hypothesis, two major investigations are necessary: (1) in the BCS animal model and clinical studies, the relationship between the severity of liver fibrosis and the expression of HIF and CD248/endosialin in HSC will be explored; and (2) in the in vitro cell system, the effect of hypoxic microenvironment, HIF-1α or HIF-2α, on the expression of CD248/endosialin in HSC will be explored. It will be important to elucidate whether HIF signaling pathway regulates the expression of CD248/endosialin, thereby inducing the development of BCS-related liver fibrosis.

Transcriptome analysis demonstrates that long noncoding RNA is involved in the hypoxic response in Larimichthys crocea.

The large yellow croaker (Larimichthys crocea) has low hypoxia tolerance compared with other fish species, and the mRNA levels of hypoxia-inducible factor (HIF)-1α in its brain do not change markedly under hypoxic conditions. In this study, we investigated noncoding transcription in the hypoxic response mechanism of L. crocea. We generated a catalog of long noncoding RNAs (lncRNAs) from the brain of L. crocea individuals under hypoxic stress, investigated lncRNA expression patterns, and analyzed the HIF signaling pathway by RNA sequencing. Prolyl hydroxylase domain 2 (PHD2) expression significantly increased after 6 and 12h of hypoxia, and a lncRNA (Linc_06633.1) was found in the upstream, antisense region of PHD2. Linc_06633.1 may be an important regulator that promotes PDH2 expression under hypoxia in L. crocea, and we constructed a regulatory profile of L. crocea under hypoxic conditions. To the best of our knowledge, it is the first study that has been conducted on hypoxia signaling pathway regulation by lncRNAs in L. crocea and elucidates the role played by lncRNAs in the regulation of the hypoxia stress response in teleost fish.

The protective effect of Hif3a RNA interference and HIF-prolyl hydroxylase inhibition on cardiomyocytes under anoxia-reoxygenation

The aim of this study was to investigate the molecular mechanisms underlying the protective effects of hypoxia-inducible factor (HIF) signaling pathway activation in cardiomyocytes under anoxia-reoxygenation (A/R) injury. In this study, rat neonatal cardiomyocytes were pretreated with anti-Hif3A/Hif-3α siRNA or HIF-prolyl hydroxylase inhibitor prior to A/R injury. Our results showed that both HIF3A silencing and HIF-prolyl hydroxylase inhibition effectively increased the cell viability during A/R, led to changes in mRNA expression of HIF1-target genes, and reduced the loss of mitochondrial membrane potential (Δψm). Furthermore, application of anti-Hif3a siRNA led to an increase in mRNA expression of Epo, Igf1, Slc2a1/Glut-1, and Slc2a4/Glut-4. Similar results were observed with HIF-prolyl hydroxylase inhibition, which additionally upregulated the mRNA expression of Epor, Tert, and Pdk1. Hif3a RNA-interference and application of HIF-prolyl hydroxylase inhibitor during A/R modelling led to an increase of Δψm on 11.5 and 11.9 mV respectively, compared to the control groups. Thus, Hif3a RNA interference and HIF-prolyl hydroxylase inhibition protect cardiomyocytes against A/R injury via the HIF signaling pathway.

Ferritin heavy chain controls the HIF-driven hypoxic response by activating the asparaginyl hydroxylase FIH

Hypoxia-inducible factor 1 (HIF-1) is a key player in cellular response to hypoxia. The stability and transcriptional activity of this protein are oxygen-dependently regulated by the prolyl hydroxylases PHD1-3 and the asparaginyl hydroxylase FIH. Recently, ferritin heavy chain (FTH1) has been characterized to reinforce the HIF-1 signaling pathway in an indirect way through the inhibition of PHD activity by depleting the free iron pool in the cytoplasm. In the present study, we addressed the role of FTH1 in the FIH control of HIF-1 activity. Unexpectedly, immunoprecipitation analyses revealed that FTH1 directly interacted with FIH. In an in vitro hydroxylation assay, FTH1 was found to facilitate the FIH-mediated Asn803 hydroxylation in HIF-1α. As expected, FTH1 prevented the recruitment of p300 to HIF-1α through the Asn803 hydroxylation. In luciferase reporter analyses, FTH1 was found to repress the transcriptional activity of HIF-1α in HCT116 cells under either normoxic or hypoxic conditions. Consequently, FTH1 downregulated the expression of the HIF-1 target genes, such as VEGF, CA9 and GLUT1. Our results suggest a new role of FTH1 as a co-regulator for the FIH-mediated oxygen sensing pathway. Since HIF-1α is involved in pathogenesis of diverse hypoxia-associated diseases, we propose that FTH1 be a potential target in developing new therapeutic strategies against such diseases.

Cadmium disrupts signaling of the hypoxia-inducible (HIF) and transforming growth factor (TGF-β) pathways in placental JEG-3 trophoblast cells via reactive oxygen species

Epidemiologic studies indicate an association between exposure to cadmium (Cd) and placental-related pregnancy disorders. While a precise mechanism is unknown, oxidative imbalance and dysregulation of the hypoxia inducible factor (HIF) and transforming growth factor beta (TGF-β) pathways have been implicated in placental disease pathogenesis. Here we investigated key oxidative and placentation pathways in JEG-3 placental trophoblast cells treated with Cd alone, environmental water samples predominated by Cd with low concentrations of other metals (e.g. inorganic arsenic (iAs)) collected from a waste-site, and a matched mixture of Cd and iAs prepared in the laboratory. The induction of cytosolic reactive oxygen species (ROS), expression of metallothionein (MT) isoforms, HIF1α and downstream targets, and expression of TGFβ pathway-associated genes and proteins were assessed. Additionally, the effect of pre-treatment with the antioxidant N-acetyl cysteine (NAC) on ROS generation and effects on HIF, MT and TGF-β signaling pathways was examined. Cd and Cd-mixture treated cells displayed higher levels of ROSs with accompanying disruption of HIF and TGFβ pathway signaling versus controls, with the Cd-mixture eliciting a greater effect. Conversely, pretreatment with NAC reduced Cd-induced ROS production and disruption of HIF, MT and TGFβ pathway signaling. The results indicate that treatment of placental trophoblast cells with Cd results in increased production of ROSs that disrupt placentation pathways involved in disease pathogenesis. Also, co-occurrence of Cd with other toxic metals, particularly arsenic, may induce detrimental health effects that are currently underestimated when analyzed as single metals.

Tumor Hypoxia As an Enhancer of Inflammation-Mediated Metastasis: Emerging Therapeutic Strategies.

Metastasis is the leading cause of cancer-related deaths. Recent research has implicated tumor inflammation as a promoter of metastasis. Myeloid, lymphoid, and mesenchymal cells in the tumor microenvironment promote inflammatory signaling amongst each other and together with cancer cells to modulate sustained inflammation, which may enhance cancer invasiveness. Tumor hypoxia, a state of reduced available oxygen present in the majority of solid tumors, acts as a prognostic factor for a worse outcome and is known to have a role in tumor inflammation through the regulation of inflammatory mediator signals in both cancer and neighboring cells in the microenvironment. Multiple methods to target tumor hypoxia have been developed and tested in clinical trials, and still more are emerging as the impacts of hypoxia become better understood. These strategies include mechanistic inhibition of the hypoxia inducible factor signaling pathway and hypoxia activated pro-drugs, leading to both anti-tumor and anti-inflammatory effects. This prompts a need for further research on the prevention of hypoxia-mediated inflammation in cancer. Hypoxia-targeting strategies seem to have the most potential for therapeutic benefit when combined with traditional chemotherapy agents. This paper will serve to summarize the role of the inflammatory response in metastasis, to discuss how hypoxia can enable or enhance inflammatory signaling, and to review established and emerging strategies to target the hypoxia-inflammation-metastasis axis.

Genome-wide gene expression changes associated with exposure of rat liver, heart, and kidney cells to endosulfan

Endosulfan was once the most commonly used pesticide in agriculture and horticulture. It is an environmentally persistent organochlorine compound with the potential to bioaccumulate as it progresses through the food chain. Its acute and chronic toxicity to mammals, including humans, is well known, but the molecular mechanisms of its toxicity are not fully understood. To gain insight to these mechanisms, we examined genome-wide gene expression changes of rat liver, heart, and kidney cells induced by endosulfan exposure. We found that among the cell types examined, kidney and liver cells were the most sensitive and most resilient, respectively, to endosulfan insult. We acquired RNA sequencing information from cells exposed to endosulfan to identify differentially expressed genes, which we further examined to determine the cellular pathways that were affected. In kidney cells, exposure to endosulfan was uniquely associated with altered expression levels of genes constituting the hypoxia-inducible factor-1 (HIF-1) signaling pathway. In heart and liver cells, exposure to endosulfan altered the expression levels of genes for many members of the extracellular matrix (ECM)-receptor interaction pathway. Because both HIF-1 signaling and ECM-receptor interaction pathways directly or indirectly control cell growth, differentiation, proliferation, and apoptosis, our findings suggest that dysregulation of these pathways is responsible for endosulfan-induced cell death.

Effects of Acanthopanax senticosus on Brain Injury Induced by Simulated Spatial Radiation in Mouse Model Based on Pharmacokinetics and Comparative Proteomics.

The active compounds in Acanthopanax senticosus (AS) have different pharmacokinetic characteristics in mouse models. Cmax and AUC of Acanthopanax senticosus polysaccharides (ASPS) were significantly reduced in radiation-injured mice, suggesting that the blood flow of mouse was blocked or slowed, due to the pathological state of ischemia and hypoxia, which are caused by radiation. In contrast, the ability of various metabolizing enzymes to inactivate, capacity of biofilm transport decrease, and lessening of renal blood flow accounts for radiation, resulting in the accumulation of syringin and eleutheroside E in the irradiated mouse. Therefore, there were higher pharmacokinetic parameters—AUC, MRT, and t1/2 of the two compounds in radiation-injured mouse, when compared with normal mouse. In order to investigate the intrinsic mechanism of AS on radiation injury, AS extract’s protective effects on brain, the main part of mouse that suffered from radiation, were explored. The function of AS extract in repressing expression changes of radiation response proteins in prefrontal cortex (PFC) of mouse brain included tubulin protein family (α-, β-tubulin subunits), dihydropyrimidinase-related protein 2 (CRMP2), γ-actin, 14-3-3 protein family (14-3-3ζ, ε), heat shock protein 90β (HSP90β), and enolase 2. The results demonstrated the AS extract had positive effects on nerve cells’ structure, adhesion, locomotion, fission, and phagocytosis, through regulating various action pathways, such as Hippo, phagosome, PI3K/Akt (phosphatidylinositol 3 kinase/protein kinase B), Neurotrophin, Rap1 (Ras-related protein RAP-1A), gap junction glycolysis/gluconeogenesis, and HIF-1 (Hypoxia-inducible factor 1) signaling pathways to maintain normal mouse neurological activity. All of the results indicated that AS may be a promising alternative medicine for the treatment of radiation injury in mouse brain. It would be tested that whether the bioactive ingredients of AS could be effective through the blood–brain barrier in the future.

Declined Presentation Inhibition of carbonic anhydrase IX as a novel strategy to target FLT3/ITD mutated aml cells under hypoxic conditions

Recent data suggests that the hypoxic properties of the bone marrow niche play a key role in the evolution of drug resistance in FLT3/ITD+AML, thus indicating a role for hypoxia-specific drugs in AML therapy. However, the mechanisms by which low oxygen (O2) levels distort drug responses of FLT3/ITD+AML cells are mostly unknown. Here we investigated the cytotoxic activity of classic and novel agents under hypoxic (1%) and normoxic (21%) conditions against FLT3/ITD+AML. Molm14 (M14) and primary cells from relapsed/refractory FLT3/ITD+AML patients were treated with Cytarabine (C), Quizartinib (Q) or FC531 (FC; a selective inhibitor of the hypoxia responsive gene carbonic anhydrase IX [CA-IX]) under 21% and 1% O2. After 48h, proliferation and apoptosis induction were assessed per MTT and FACS assays. Cells were also assessed for FLT3 protein and Hypoxia inducible factor (HIF) target gene expression by western blot and PCR arrays. The growth inhibitory effects of C and Q against FLT3/ITD+ cells were significantly dampened under 1% compared to 21% O2. Similarly, hypoxia weakened the pro-apoptotic effects of either drug. In contrast, Q potently inhibited FLT3 activity under both 1% and 21% O2. We next sought to identify tractable pro-survival genes induced by hypoxia, and to assess their expression in response to therapy. Our data shows that the HIF pathway is upregulated in response to 1% O2 and remains widely functional in C or Q treated cells. CA-IX, a druggable HIF target, was significantly upregulated in FLT3/ITD+ cells under 1% O2. When we exposed M14 cells to FC, we found that cell growth was significantly inhibited under 1% but not 21% O2. Further, FC was significantly more effective than C or Q in inducing apoptosis under hypoxia. 1) Hypoxia blunts the cytotoxic effects of C and Q in FLT3/ITD+AML cells. 2) The HIF signaling pathway remains largely functional under hypoxic conditions, despite treatment. 3) FC is significantly more cytotoxic than C or Q under hypoxic conditions. 4) Further investigation of combined therapies between standard AML agents and FC in FLT3/ITD+AML is warranted.

18F-FDG Uptake in Well-Differentiated Neuroendocrine Tumors Correlates with Both Ki-67 and VHL Pathway Inactivation

Background:¹⁸F-FDG-PET scan positivity correlates with poor prognosis in neuroendocrine neoplasms (NEN). Glucose transporter 1 (GLUT1) and carbonic anhydrase 9 (CA9) are markers of aggressiveness in tumors. Together with von Hippel-Lindau protein (pVHL), they are involved in tumor cell metabolism via the hypoxia-inducible factor signaling pathway. The aim of this study was to compare, in a series of well-differentiated neuroendocrine tumors (NET), the ¹⁸F-FDG uptake and expression of the proliferation markers Ki-67, GLUT1, CA9, and pVHL. Patients and Methods: This retrospective study included 27 patients with well-differentiated NET. ¹⁸F-FDG-PET images were evaluated by the maximum standardized uptake value (SUV_max). GLUT1, CA9, and pVHL were analyzed by immunohistochemistry. Results: The NET were of pancreatic (n = 19), midgut (n = 4), duodenal (n = 1), esophageal (n = 1), rectal (n = 1), and pulmonary (n = 1) origin. Eight, 11, and 8 tumors were grade 1, 2, and 3, respectively. The mean/median Ki-67 index was 15/10% (1-60). The mean/median SUV_max was 6.2/5.2 (1.4-18.7). SUV_max correlated with greater tumor size (p = 0.03), higher expression of Ki-67 (p = 0.04), and lower expression of pVHL (p = 0.008). In the group of 16 NET with a low proliferative index (Ki-67 index <10%), 5/6 (83%) of the tumors with a high SUV_max had decreased pVHL expression (p = 0.0013). Conclusion: This study confirms that ¹⁸F-FDG-PET uptake correlates with both tumor size and proliferation in well-differentiated NET, and it highlights a subset of low-grade but ¹⁸F-FDG-PET-positive NET related to sporadic inactivation of the VHL pathway.

Cooperation of HIF- and NCAM-mediated mechanisms in cell viability of hippocampal cultures after oxygen-glucose deprivation.

Neurodegenerative diseases of different genesis are the result of cellular damages including those caused by oxygen and glucose deficit. Neuronal survival or death in brain pathologies depends on a variety of interrelated molecular mechanisms. A key role in modulation of neuron viability belongs to HIF (hypoxia-inducible factor) and NCAM (neural cell adhesion molecules) signaling pathways. In this work, we used organotypic and dissociated hippocampal cultures to analyze cell viability and HIF-1α immunopositive (HIF-1α+ ) signal after 30 min oxygen-glucose deprivation (OGD) followed by 24 h of reoxygenation in the presence of FGL (synthetic NCAM-derived mimetic peptide). According to LDH- and MTS-assay of cell viability, FGL showed a neuroprotective effect, which was attributed to the association with FGFR. We showed that these effects correlated with changes of the HIF-1α+ level suggesting the communications of HIF and NCAM signaling pathways. These data extend our knowledge of neurodegeneration mechanisms and open additional potential for the development of neuroprotection strategies.

Network-based analysis of the molecular mechanisms of multiple myeloma and monoclonal gammopathy of undetermined significance.

The present study aimed to reveal the molecular mechanisms of multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). This was a secondary study on microarray dataset GSE80608, downloaded from the Gene Expression Omnibus database, which included 10 control samples, 10 MGUS samples and 10 MM samples. Differentially expressed genes (DEGs) were identified between control and MGUS samples, and between control and MM samples. A protein-protein interaction (PPI) network was built for studying the interactions between the DEGs. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed for the genes in a gene co-expression network. A microRNA (miRNA/miR)-gene network was built to the evaluate possible the miRNAs and genes involved in the diseases. The present study identified 136 common upregulated DEGs and 165 common downregulated DEGs between MM and MGUS. Pathway enrichment analysis of the genes in the gene co-expression network revealed that the complement and coagulation cascades pathway was significantly enriched for certain complement and coagulation-associated genes. Endothelin-1 (EDN1) was significantly enriched in the hypoxia inducible factor-1 (HIF-1) and tumor necrosis factor signaling pathways. EDN1 was an important node in the PPI network, and a target gene of let-7e, let-7b and miR-19a in the miRNA-gene network. The results of the present study indicate that complement and coagulation-associated genes, the complement and coagulation cascades pathway, EDN1, let-7e, let-7b-5p, miR-19a, and the tumor necrosis factor and HIF-1 signaling pathways may all be implicated in MM and MGUS.

Prolyl Hydroxylase Domain-Containing Protein 2 (Phd2) Regulates Chondrocyte Differentiation and Secondary Ossification in Mice.

Endochondral ossification plays an important role in the formation of the primary ossification centers (POCs) and secondary ossification centers (SOCs) of mammalian long bones. However, the molecular mechanisms that regulate POC and SOC formation are different. We recently demonstrated that Prolyl Hydroxylase Domain-containing Protein 2 (Phd2) is a key mediator of vitamin C effects on bone. We investigated the role of Phd2 on endochondral ossification of the epiphyses by conditionally deleting the Phd2 gene in osteoblasts and chondrocytes. We found that the deletion of Phd2 in osteoblasts did not cause changes in bone parameters in the proximal tibial epiphyses in 5 week old mice. In contrast, deletion of Phd2 in chondrocytes resulted in increased bone mass and bone formation rate (normalized to tissue volume) in long bone epiphyses, indicating that Phd2 expressed in chondrocytes, but not osteoblasts, negatively regulates secondary ossification of epiphyses. Phd2 deletion in chondrocytes elevated mRNA expression of hypoxia-inducible factor (HIF) signaling molecules including Hif-1α, Hif-2α, Vegfa, Vegfb, and Epo, as well as markers for chondrocyte hypertrophy and mineralization such as Col10, osterix, alkaline phosphatase, and bone sialoprotein. These data suggest that Phd2 expressed in chondrocytes inhibits endochondral ossification at the epiphysis by suppressing HIF signaling pathways.

Silencing of hypoxia inducible factor-1α gene attenuated angiotensin Ⅱ-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice

Background and aimsWe aimed to determine the effect of HIF-1α, the main regulatory subunit of the hypoxia inducible factor 1 (HIF-1), on the development of the abdominal aortic aneurysm (AAA). MethodsAAA was induced in ApoE−/− mice by angiotensinⅡ (AngⅡ) infusion. In vivo silencing of HIF-1α was achieved by transfection of lentivirus expressing HIF-1α shRNA. ResultsTime course analysis of the AngⅡ infusion model revealed that HIF-1α was persistently upregulated during a 28-day period of AAA development. Silencing of the HIF-1α gene reduced the aneurysm size (2.84 ± 1.96 mm vs. 1.41 ± 0.85 mm respectively at day 28, p = 0.0002). Silencing of HIF-1α also alleviated infiltration of macrophages (38.8 ± 14.7 vs. 11.4 ± 4.4 macrophages/0.1 mm2, p = 0.0006) and neovascularity (5.56 ± 2.14 vs. 1.27 ± 1.05 microvessels/0.1 mm2, p = 0.0008) in the AngⅡ infusion model, at day 28. The activity of MMP-2 and MMP-9 was also decreased by knockdown of HIF-1α. The early increased expression of pro-inflammatory factors, angiogenic factors, and MMPs during AAA induction was alleviated by HIF-1α silencing. ConclusionsActivation of HIF-1 signaling pathway participates in the Ang Ⅱ-induced AAA formation in mice.

Abstract 3273: The efficiency of anti-tumor effect of metformin for gemcitabine-resistant pancreatic adenocarcinoma

Abstract Metformin (MET) is the first-line treatment for type 2 diabetes mellitus. Several epidemiological studies have revealed the anti-cancer effects of metformin (MET), including against pancreatic ductal carcinoma (PDC). Gemcitabine (GEM) has become the standard chemotherapy for PDC but tolerance to GEM becomes an important issue. We evaluated the anti-tumor effects of MET for GEM-resistant PDC in a xenograft mouse model. For this in vivo study, wild-type BxPC-3 was implanted into both flanks of female BALB/c nude mice. Mice were divided into four groups: (i) control (without any treatment); (ii) GEM-treated group (100 mg/kg); (iii) MET-treated group (600 mg/kg); and (iv) combined treatment group (G+M). Mice were fed for 4 weeks. Estimated tumor volumes and body weights were measured each week. Treatments were initiated 2 weeks after implantation. MET was administrated orally every day. GEM was given by intraperitoneal injection every week. In the final tumor volumes, the two groups treated with GEM had significantly reduced tumors compared with control, but there were no differences between control and MET groups. The anti-tumor effect of MET for BxG30 (the cell line for GEM-resistant PDC) was evaluated using the method described above. Tumor volumes were decreased significantly in GEM+MET groups compared with control. The treated control ratio (T/C%) was calculated: GEM, 80.2%; MET, 54.0%; GEM+MET, 47.2%. The anti-tumor effect of GEM for BxG30 was limited. The MET group showed satisfactory anti-tumor effects, but T/C% was &amp;lt;50% only in the GEM+MET group. This result revealed that combination therapy had excellent anti-tumor effects even for GEM-resistant PDC. Western blot analysis was performed to confirm mammalian target of rapamycin (mTOR) expression level was surely inhibited. The results revealed that the expression level of mTOR treated with MET was significantly decreased, but no in GEM group. The expression level of hypoxia-inducible factor 1 (HIF-1) was evaluated by western blot analysis also. The results showed significant inhibition of HIF-1 expression by MET treatment, but not by GEM, again. Then the expression of VEGF mRNA, downstream of HIF-1 signaling pathway, was evaluated by qRT-PCR. The result showed inhibition of VEGF mRNA expression by MET treatment. Our results showed that MET has a partial anti-tumor effect for PDC. Combination therapy has an excellent effect not only for wild-type PDC but also for GEM-resistant PDC. The mechanism of anti-tumor effect of MET seems to be, at least partly, the inhibition of HIF-1/mTOR signaling pathway. These data suggest that MET could be used to treat PDC. Citation Format: Keiichi Suzuki, Osamu Takeuchi, Yoshiyuki Ishii, Masayoshi Osaku. The efficiency of anti-tumor effect of metformin for gemcitabine-resistant pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3273.

AMPK and HIF signaling pathways regulate both longevity and cancer growth: the good news and the bad news about survival mechanisms.

The AMP-activated protein kinase (AMPK) and hypoxia-inducible factor (HIF) signaling pathways are evolutionarily-conserved survival mechanisms responding to two fundamental stresses, energy deficiency and/or oxygen deprivation. The AMPK and HIF pathways regulate the function of a survival network with several transcription factors, e.g. FOXO, NF-κB, NRF2, and p53, as well as with protein kinases and other factors, such as mTOR, ULK1, HDAC5, and SIRT1. Given that AMPK and HIF activation can enhance not only healthspan and lifespan but also cancer growth in a context-dependent manner; it seems that cancer cells can hijack certain survival factors to maintain their growth in harsh conditions. AMPK activation improves energy metabolism, stimulates autophagy, and inhibits inflammation, whereas HIF-1α increases angiogenesis and helps cells to adapt to severe conditions. First we will review how AMPK and HIF signaling mechanisms control the function of an integrated survival network which is able not only to improve the regulation of longevity but also support the progression of tumorigenesis. We will also describe distinct crossroads between the regulation of longevity and cancer, e.g. specific regulation through the AMPKα and HIF-α isoforms, the Warburg effect, mitochondrial dynamics, and cellular senescence.

Hypoxia Suppressed Copper Toxicity during Early Development in Zebrafish Embryos in a Process Mediated by the Activation of the HIF Signaling Pathway.

Hypoxia is a global and increasingly important stressor in aquatic ecosystems, with major impacts on biodiversity worldwide. Hypoxic waters are often contaminated with a wide range of chemicals but little is known about the interactions between these stressors. We investigated the effects of hypoxia on the responses of zebrafish (Danio rerio) embryos to copper, a widespread aquatic contaminant. We showed that during continuous exposures copper toxicity was reduced by over 2-fold under hypoxia compared to normoxia. When exposures were conducted during 24 h windows, hypoxia reduced copper toxicity during early development and increased its toxicity in hatched larvae. To investigate the role of the hypoxia signaling pathway on the suppression of copper toxicity during early development, we stabilized the hypoxia inducible factor (HIF) pathway under normoxia using a prolyl-4-hydroxylase inhibitor, dimethyloxalylglycine (DMOG) and demonstrated that HIF activation results in a strong reduction in copper toxicity. We also established that the reduction in copper toxicity during early development was independent of copper uptake, while after hatching, copper uptake was increased under hypoxia, corresponding to an increase in copper toxicity. These findings change our understanding of the current and future impacts of worldwide oxygen depletion on fish communities challenged by anthropogenic toxicants.