HIF-1α Protein Levels Research Articles

Intravitreal Administration of AAV2-SIRT1 Reverses Diabetic Retinopathy in a Mouse Model of Type 2 Diabetes.

The expression of silent information regulator (SIRT) 1 is reduced in diabetic retinopathy (DR). Previous studies showed that alterations in SIRT1 messenger RNA (mRNA) and protein expression are implicated in progressive inflammation and formation of retinal acellular capillaries. Treatment with the SIRT1 agonist, SRT1720, improved visual response by restoration of a- and b-wave responses on electroretinogram scotopic measurements in diabetic (db/db) mice. In this study, we investigated the effects of intravitreal SIRT1 delivery on diabetic retinal pathology. Nine-month-old db/db mice received one intravitreal injection of either AAV2-SIRT1 or AAV2-GFP control virus, and after 3 months, electroretinography and optomotor responses were measured. Their eyes were then removed and analyzed by immunohistochemistry and flow cytometry. SIRT1 mRNA and protein levels were increased following AAV2-SIRT1 administration compared to control virus AAV2-GFP injected mice. IBA1+ and caspase 3 expression were decreased in retinas of db/db mice injected with AAV2-SIRT1, and reductions in scotopic a- and b-waves and high spatial frequency in optokinetic response were prevented. Retinal hypoxia inducible factor 1α (HIF-1α) protein levels were reduced in the AAV2-SIRT1-injected mice compared to control-injected mice. Using flow cytometry to assess changes in intracellular HIF-1α levels, endothelial cells (CD31+) from AAV-2 SIRT1 injected mice demonstrated reduced HIF-1α expression compared to db/db mice injected with the control virus. Intravitreal AAV2-SIRT1 delivery increased retina SIRT1 and transduced neural and endothelial cells, thus reversing functional damage and improving overall visual function. AAV2-SIRT1 gene therapy represents a beneficial approach for the treatment of chronic retinal conditions such as DR.

Hypoxia-inducible lncRNA MIR210HG promotes HIF1α expression by inhibiting miR-93-5p in renal tubular cells.

Chronic hypoxia in the renal tubular interstitium has been reported to contribute to the progression of chronic kidney disease. Recently, long-noncoding RNAs have been shown to be involved in various pathological conditions, including hypoxia, one of which is the MIR210 host gene (MIR210HG). To elucidate the function of MIR210HG in renal hypoxia, we exposed primary cultured renal proximal tubular epithelial cells to hypoxia and examined the temporal profile of MIR210HG expression and the role of MIR210HG interaction with hypoxia-inducible factor1α (HIF1α, encoded by HIF1A). MIR210HG expression was induced by hypoxia. HIF1A silencing and cobalt chloride exposure showed that MIR210HG expression in hypoxia is HIF1α-dependent. MIR210HG silencing significantly reduced both the mRNA and protein levels of HIF1α, pointing to positive feedback regulation. To further investigate the details of this regulation, we turned to the in-silico miRNA targets of MIR210HG. We found that miR-93-5p levels increased when MIR210HG was knocked down. We then showed that miR-93-5p reduced the expression of HIF1A mRNA and MIR210HG. Furthermore, a dual luciferase assay confirmed that miR-93-5p binds to MIR210HG and HIF1A 3' UTR, inhibiting their expression. In conclusion, the long-noncoding RNA MIR210HG is induced shortly after hypoxia, and it promotes HIF1α expression by competing for miR-93-5p and inhibiting it. MIR210HG plays a crucial role in the biological response to hypoxia in renal tubular epithelial cells.

ABL kinases regulate the stabilization of HIF-1α and MYC through CPSF1

The hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx. We show that ABL kinases phosphorylate and interact with CUL4A, a cullin ring ligase adaptor, and compete with CPSF1 for CUL4A binding, leading to increased HIF-1α protein levels. Further, we identified the MYC proto-oncogene protein as a second CPSF1 substrate and show that active ABL kinase protects MYC from CPSF1-mediated degradation. These studies uncover a role for CPSF1 in cancer pathobiology as an E3-ligase antagonizing the expression of the oncogenic transcription factors, HIF-1α and MYC.

Differential Impact of Intermittent vs. Sustained Hypoxia on HIF-1, VEGF and Proliferation of HepG2 Cells.

Obstructive sleep apnea (OSA) is an emerging risk factor for cancer occurrence and progression, mainly mediated by intermittent hypoxia (IH). Systemic IH, a main landmark of OSA, and local sustained hypoxia (SH), a classical feature at the core of tumors, may act separately or synergistically on tumor cells. Our aim was to compare the respective consequences of intermittent and sustained hypoxia on HIF-1, endothelin-1 and VEGF expression and on cell proliferation and migration in HepG2 liver tumor cells. Wound healing, spheroid expansion, proliferation and migration were evaluated in HepG2 cells following IH or SH exposure. The HIF-1α, endothelin-1 and VEGF protein levels and/or mRNA expression were assessed, as were the effects of HIF-1 (acriflavine), endothelin-1 (macitentan) and VEGF (pazopanib) inhibition. Both SH and IH stimulated wound healing, spheroid expansion and proliferation of HepG2 cells. HIF-1 and VEGF, but not endothelin-1, expression increased with IH exposure but not with SH exposure. Acriflavine prevented the effects of both IH and SH, and pazopanib blocked those of IH but not those of SH. Macitentan had no impact. Thus, IH and SH stimulate hepatic cancer cell proliferation via distinct signaling pathways that may act synergistically in OSA patients with cancer, leading to enhanced tumor progression.

Identification and clinical validation of hypoxia-inducible factor -1α protein as the potential biomarker in patients with sepsis.

Sepsis is a complex disease characterized by an inflammatory response and tissue hypoxia. Hypoxia-inducible factor-1α (HIF-1α) expression level is regulated by hypoxia and inflammation. This study aimed to explore the correlation between HIF-1α expression level and sepsis by bioinformatics analysis and clinical investigation. Bioinformatics tools were used to identify differentially expressed genes (DEGs) between sepsis and non-sepsis groups using the Gene Expression Omnibus (GEO) dataset. A clinical investigation was carried out to validate HIF-1α protein level in 54 non-septic patients and 173 septic patients who were followed up for 28 days. Bioinformatics analysis revealed that HIF-1α mRNA level was significantly different between septic and non-septic patients (P < 0.05). Consistent with the study hypothesis, higher HIF-1α levels in plasma were found in septic patients compared with those in non-septic patients. The diagnostic accuracy for sepsis, as quantified by the area under the curve, was 0.926 (0.885-0.968) for HIF-1α expression level combined with oxygen saturation to fraction of inspired oxygen (SpO2/ FiO2), white blood cell (WBC) and blood urea nitrogen (BUN). The HIF-1α expression level was also significantly correlated with the severity of the disease. The results of the restricted cubic splines (RCS) model indicated a U-shaped relationship between HIF-1α expression level and intensive care unit (ICU) mortality. Univariate and multivariate linear regression analyses indicated that septic patients with the elevated HIF-1α expression levels had shorter length of ICU stay versus those with the lower HIF-1α expression levels. HIF-1α expression level can be used for diagnosing disease, assessing severity, and predicting length of ICU stay in septic patients.

A cell-permeable peptide inhibitor of p55PIK signaling alleviates suture-induced corneal neovascularization and inflammation

To prepare an ophthalmic solution with a cell-permeable TAT peptide (TAT-N24) as the main cell-permeable peptide inhibitor of p55PIK signaling and observe its therapeutic effect on suture-induced corneal neovascularization (CNV) in rats. Sprague-Dawley rats were used to establish a corneal suture (CS) model of CNV. The vehicle and 0.9% TAT-N24 ophthalmic solution was topically administered. CNV induction was assessed on the basis of the clinical performance of each group. Hematoxylin-eosin staining was used to observe pathological changes, and immunohistochemical staining and confocal immunofluorescence were used to determine the localization of factors associated with corneal tissue. The mRNA expression levels of hypoxia-inducible factor (HIF-1α), vascular endothelial growth factor (VEGF-A), nuclear transcription factor κB (NF-κB p65), tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and interleukin (IL)-6 were determined using real-time quantitative polymerase chain reaction. Western blotting was performed to detect the protein expression levels of HIF-1α and NF-κB p65. TAT-N24 slowed CNV production and reduced the expression of HIF-1α and inflammatory factors in CS models. The mRNA levels of HIF-1α, VEGF-A, NF-kB, TNF-α, IL-1β, and IL-6 significantly decreased. Moreover, the protein levels of HIF-1α and NF-κB p65 were significantly decreased. TAT-N24 can treat CNV and ocular inflammation by inhibiting the HIF-1α/NF-κB signaling pathway in CS. In the early treatment of corneal foreign body trauma, topical application of TAT-N24 can not only reduce the inflammatory response but also inhibit corneal neovascularization.

Clinical and pathological features analysis of invasive breast cancer with microcalcification.

Microcalcification (MC) is a valuable diagnostic indicator to detect invasive breast cancer (IBC). This study aimed to determine the clinicopathological features of IBC with MC and detect biomarkers related to the potential mechanism of the MC formation in IBC. Data from 364 patients with IBC were collected for the clinical characteristic analysis. The analysis of clinical data helped us to establish a predictive model of axillary node metastasis (ANM) before surgery. In addition, 49 tissue samples of IBC patients were collected to test the protein levels of osteocalcin (OCN) and hypoxia-inducible factor-1α (HIF-1α) by immunohistochemistry. Significant differences were observed in tumor size, age, ANM, HER2+ , TNM stage, and mutant P53 between samples from IBC patients with MC and samples from IBC patients without MC. Younger age, a larger tumor size, a higher number of childbirths, and MC were independent predictors for ANM in IBC. HIF-1α protein level was higher in tumor tissue than in normal tissue. High protein levels of OCN and HIF-1α are related to the complication of MC in IBC. Of the patients that exhibited high HIF-1α protein levels, the percentage of high OCN protein levels was larger in patients with ANM. Based on this study, we concluded that patients with MC had a comparatively poor prognosis. MC was an independent predictive factor associated with the risk of ANM. High protein levels of OCN and HIF-1α were associated with MC and ANM, which were also related to poor prognosis. OCN and HIF-1α had a positive correlation in IBC.

Could Local Application of Hypoxia Inducible Factor 1-α Enhancer Deferoxamine Be Promising for Preventing of Medication-Related Osteonecrosis of the Jaw?

This experimental study investigates the prophylactic effect of deferoxamine (DFO) on medication-related osteonecrosis of the jaw (MRONJ). Thirty-six female Sprague Dawley rats received zoledronic acid (ZA) for eight weeks to create an osteonecrosis model. DFO was locally applied into the extraction sockets with gelatin sponge (GS) carriers to prevent MRONJ. The specimens were histopathologically and histomorphometrically evaluated. Hypoxia-inducible factor 1-alpha (HIF-1α) protein levels in the extraction sockets were quantified. New bone formation rate differed significantly between groups (p = 0.005). Newly formed bone ratios in the extraction sockets did not differ significantly between the control group and the GS (p = 1), GS/DFO (p = 0.749), ZA (p = 0.105), ZA-GS (p = 0.474), and ZA-GS/DFO (p = 1) groups. While newly formed bone rates were higher in the ZA-GS and ZA-GS/DFO groups than in the ZA group, the differences were not significant. HIF-1α levels differed significantly between groups (p < 0.001) and were significantly higher in the DFO and ZA-GS/DFO groups than in the control group (p = 0.001 and p = 0.004, respectively). While HIF-1α levels were higher in the ZA-GS/DFO group than in the ZA group, the difference was not significant. While HIF-1α protein levels and new bone formation rate were elevated in the DFO-treated group, the effect was not significant. Further large-scale studies are needed to understand DFO's preventative effects on MRONJ and the role of HIF-1α in MRONJ pathogenesis.

FtMt reduces oxidative stress-induced trophoblast cell dysfunction via the HIF-1α/VEGF signaling pathway

BackgroundPreeclampsia (PE) is a complication of pregnancy that causes long-term adverse outcomes for the mother and fetus and may even lead to death. Oxidative stress caused by the imbalance of oxidants and antioxidants in the placenta has been considered as one of the key mechanisms of preeclampsia (together with inflammation, etc.), in which the placental mitochondria play an important role. The expression of hypoxia-inducible factor-1 (HIF-1α) and vascular endothelial growth factor (VEGF) is known to be increased in patients with PE. Mitochondrial ferritin (FtMt) is known to protect the mitochondria from oxidative stress, although its specific role in PE remains unclear.MethodsWe used qRT-PCR and western blotting to detect the expression levels of FtMt, HIF-1α, and VEGF in placental tissues from patients with PE. Human chorionic trophoblast cells were also administered with hypoxia treatment, followed by the detection of cell proliferation, invasion and angiogenic capacity by CCK8, Transwell, and endothelial cell angiogenesis assays; we also detected the expression of HIF-1α and VEGF in these cells. Finally, overexpression or inhibitory FtMt lentiviral vectors, along with negative control vectors, were constructed and transfected into hypoxia-treated human chorionic trophoblast cells; this was followed by analyses of cell function.ResultsThe expression levels of FtMt, HIF-1α and VEGF in the PE group were higher than those in the control group (P < 0.05). Following hypoxia, there was an increase in the expression levels of HIF-1α and VEGF protein in trophoblast cells. There was also an increase in invasion ability and vascular formation ability along with a reduction in cell proliferation ability. These effects were reversed by transfecting cells with the knockout FtMt lentivirus vector. The differences were statistically significant.ConclusionAnalyses showed that FtMt plays a key role in the vascular regulation of PE trophoblast cells after hypoxia possibly acting via the HIF-1α/VEGF signaling pathway. These results provide us an enhanced understanding of the pathogenesis of PE and suggest that the HIF-1α/VEGF signaling pathway represents a new target for the treatment of PE.

LncRNA FRMD6-AS1 promotes hepatocellular carcinoma cell migration and stemness by regulating SENP1/HIF-1α axis.

Long non-cording RNAs (lncRNAs) drive the malignant progression of hepatocellular carcinoma (HCC), a cancer with high mortality rates but the function of FERM Domain Containing 6 antisense RNA 1 (FRMD6-AS1) in HCC has not been fully addressed. Hypoxia-inducible factors (HIFs) are transcription factors relevant to HCC under hypoxia and are regulated by SUMO-specific protease 1 (SENP1) through its deSUMOylation of HIF-1α. The current study investigated the role of FRMD6-AS1 in the regulation of SENP1-mediated deSUMOylation of HIF-1α. HUH7 and MHCC97H cells were treated with CoCl2 to mimic hypoxia in vitro and lentiviral vector-mediated FRMD6-AS1 overexpressing HCC cells were established. Wound-healing, Transwell, sphere formation assay, Western blotting analysis and animal experiments were performed. Expression of FRMD6-AS1, SENP1 mRNA and HIF-1α mRNA was assessed by RT-qPCR and of HIF-1α and SENP1 protein by Western blot. DeSUMOylation of HIF-1α was detected by immunoprecipitation. RNA immunoprecipitation with SENP1 antibody or IgG was performed to assess endogenous interactions between SENP1 and FRMD6-AS1. FRMD6-AS1 was upregulated in HCC tissues and cells and its upregulation indicated poor prognosis for HCC patients. FRMD6-AS1 promoted HCC cells migration and stemness in vitro and also promoted tumor growth in an in vivo mouse xenograft model. Mechanistic studies showed that FRMD6-AS1 regulated the level of HIF-1α protein but not the mRNA and this effect was achieved by binding to SENP1 protein and enhancing its protease activity. Rescue experiments demonstrated the oncogenic role of the FRMD6-AS1/SENP1/ HIF-1α axis in HCC cells. High FRMD6-AS1 expression was associated with poor prognosis of HCC patients. FRMD6-AS1 may have an oncogenic role in HCC via regulation of the SENP1/HIF-1α axis and may be a prognostic biomarker for HCC. Blockade of FRMD6-AS1 may offer a novel therapeutic approach to restrict HCC progression.

Abstract P4-08-23: ZHX2 promotes HIF1α oncogenic signaling in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease in women, while the oncogenic mechanisms which contribute to TNBC development, progression, and metastasis are poorly investigated and therefore warrant the critical need to identify novel oncogenic signalings and develop new therapeutic targets. Results: We show that Zinc Fingers and Homeoboxes 2 (ZHX2) is amplified or overexpressed in TNBC cell lines and patients. Functionally, depletion of ZHX2 inhibited TNBC cell growth and invasion in vitro, orthotopic tumor growth, and spontaneous lung metastasis in vivo. Mechanistically, ZHX2 bound with hypoxia-inducible factor (HIF) family members and positively regulated HIF1α protein level and activity in TNBC. Integrated ChIP-seq and gene expression profiling demonstrated that ZHX2 co-occupied with HIF1α on transcriptionally active promoters marked by H3K4me3 and H3K27ac, thereby promoting gene expression. Among the identified ZHX2 and HIF1α coregulated genes, overexpression of AP2B1, COX20, KDM3A, or PTGES3L could partially rescue TNBC cell growth defect by ZHX2 depletion, suggesting that these downstream targets contribute to the oncogenic role of ZHX2 in an accumulative fashion. Furthermore, multiple residues (R491, R581, and R674) on ZHX2 are important in regulating its phenotype, which correspond with their roles in controlling ZHX2 transcriptional activity in TNBC cells. These studies establish that ZHX2 activates oncogenic HIF1α signaling, therefore serving as a potential therapeutic target for TNBC. Conclusion: ZHX2 has been highlighted as one critical hypoxia-related factors regulator contributing to triple-negative breast cancer in this work, which has enriched the upstream regulatory network of HIF-1α signaling. Moreover, identification of key residuals determining biological function of ZHX2 provides novel approaches for treating TNBC via targeting the hypoxia pathway. Citation Format: Chengheng Liao, Wentong Fang, Rachel Shi, Jeremy Simon, Travis Ptacek, Giada Zurlo, Youqiong Ye, Leng Han, Cheng Fan, Lei Bao, Christopher Llynard Ortiz, Hong-Rui Lin, Ujjawal Manocha, Weibo Luo, Yan Peng, William Y Kim, Lee-Wei Yang, Qing Zhang. ZHX2 promotes HIF1α oncogenic signaling in triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-23.

Hypoxia signaling in the equine small intestine: Expression and distribution of hypoxia inducible factors during experimental ischemia.

Hypoxia inducible factors (HIF) are widely researched in human medicine for their role in different disease processes. The aim of this study was to investigate the expression and distribution of HIF in experimental small intestinal ischemia in the horse. In 14 horses under general anesthesia, segmental jejunal ischemia with 90% reduction in blood flow was induced. The horses were randomly divided into two groups of seven horses, one subjected to ischemic postconditioning (IPoC) by delayed reperfusion, and a control group (group C) undergoing undelayed reperfusion. Intestinal samples were taken pre-ischemia, after ischemia and after reperfusion. Following immunohistochemical staining for HIF1α and -2α, the immunoreactivity pattern in the small intestine was evaluated by light microscopy, and the mucosal enterocyte and muscularis staining were semi-quantitatively scored. Additionally, mucosal HIF1α protein levels were determined by an Enzyme Linked Immunosorbent Assay (ELISA), and mRNA levels of HIF1α and its target genes by a two-step real-time Reverse Transcriptase Polymerase Chain Reaction. Statistical comparison was performed between the groups and time points using parametric and non-parametric tests (p < 0.05). All cell types exhibited cytoplasmic and nuclear immunoreactivity for HIF1α. After reperfusion, the cytoplasmic staining of the crypt and villus enterocytes as well as the villus nuclear staining significantly increased, whereas the perinuclear granules in the crypts decreased. The protein levels showed a significant decrease in group C at reperfusion, with lower HIF1α levels in group C compared to group IPoC during ischemia and reperfusion. No other group differences could be detected. In the HIF2α stained slides, mild to moderate cytoplasmic staining yet no nuclear immunoreactivity of the enterocytes was observed, and no significant changes over time were noted. the changes in HIF1α immunoreactivity pattern and expression over time suggest that this transcription factor plays a role in the intestinal response to ischemia in horses. However, the current study could not identify an effect of IPoC on HIF distribution or expression.

Molecular Aspects of Hypoxic Stress Effects in Chronic Ethanol Exposure of Neuronal Cells

Experimental models of a clinical, pathophysiological context are used to understand molecular mechanisms and develop novel therapies. Previous studies revealed better outcomes for spinal cord injury chronic ethanol-consuming patients. This study evaluated cellular and molecular changes in a model mimicking spinal cord injury (hypoxic stress induced by treatment with deferoxamine or cobalt chloride) in chronic ethanol-consuming patients (ethanol-exposed neural cultures (SK-N-SH)) in order to explain the clinical paradigm of better outcomes for spinal cord injury chronic ethanol-consuming patients. The results show that long-term ethanol exposure has a cytotoxic effect, inducing apoptosis. At 24 h after the induction of hypoxic stress (by deferoxamine or cobalt chloride treatments), reduced ROS in long-term ethanol-exposed SK-N-SH cells was observed, which might be due to an adaptation to stressful conditions. In addition, the HIF-1α protein level was increased after hypoxic treatment of long-term ethanol-exposed cells, inducing fluctuations in its target metabolic enzymes proportionally with treatment intensity. The wound healing assay demonstrated that the cells recovered after stress conditions, showing that the ethanol-exposed cells that passed the acute step had the same proliferation profile as the cells unexposed to ethanol. Deferoxamine-treated cells displayed higher proliferative activity than the control cells in the proliferation-migration assay, emphasizing the neuroprotective effect. Cells have overcome the critical point of the alcohol-induced traumatic impact and adapted to ethanol (a chronic phenomenon), sustaining the regeneration process. However, further experiments are needed to ensure recovery efficiency is more effective in chronic ethanol exposure.

Oncolytic Avian Reovirus σA-Modulated Upregulation of the HIF-1α/C-myc/glut1 Pathway to Produce More Energy in Different Cancer Cell Lines Benefiting Virus Replication.

Our previous reports proved that the structural protein σA of avian reovirus (ARV) is an energy activator which can regulate cellular metabolism that is essential for virus replication. This study has further demonstrated that the ARV protein σA is able to upregulate the HIF-1α/myc/glut1 pathway in three cancer cell lines (A549, B16-F10, and HeLa) to alter the metabolic pathway of host cells. Quantitative real-time RT-PCR and Western blotting results have revealed that σA protein could enhance both mRNA and the protein levels of HIF-1α, c-myc, and glut1 in these cancer cell lines. In this work, ATeam immunofluorescence staining was used to reveal that knockdown of HIF-1α, c-myc, and glut1 by shRNAs decreased cellular ATP levels. Our data reveal that the ARV σA protein can downregulate lactate fermentation and upregulate glutaminolysis. The σA protein upregulates glutaminase, which converts glutamate into the TCA cycle intermediate α-ketoglutarate, activating the TCA cycle. In the lactate fermentation pathway, ARV σA protein suppresses lactate dehydrogenase A (LDHA), implying the Warburg effect does not occur in these cancer cell lines. This study provides a novel finding revealing that ARV σA protein upregulates glycolysis and glutaminolysis to produce energy using the HIF-1α/c-myc/glut1 pathway to benefit virus replication in these cancer cell lines.

125I seed implantation enhances arsenic trioxide-induced apoptosis and anti-angiogenesis in lung cancer xenograft mice.

Arsenic trioxide (ATO) exerts anticancer effects on lung cancer. However, the clinical use of ATO is limited due to its systemic toxicity and resistance of lung cancer cells. The present study aimed to investigate the effects of ATO, alone and in combination with 125I seed implantation on tumor growth and proliferation in lung cancer xenograft mice, and investigate the possible molecular mechanisms. The transmission electron microscope observed the tumor ultrastructure of lung cancer xenograft mice. The proliferation index of Ki-67 and the number and morphology of tumor microvessels were detected with immunohistochemical staining. The protein and mRNA expression were examined by western blot and real-time PCR assay. The in vivo results demonstrated that ATO combined with 125I seed significantly inhibited tumor growth and proliferation, as well as promoted apoptosis, and decreased the Ki-67 index and microvessel density in lung cancer xenograft mice. Moreover, ATO combined with 125I seed decreased the protein and mRNA expression levels of HIF-1α, VEGF, and BCL-2, and increased those of BAX and P53. ATO combined with 125I seed significantly inhibited tumor growth and proliferation in lung cancer, which may be accomplished by inhibiting tumor angiogenesis and inducing apoptosis.

Intraoperative oxygen tension and redox homeostasis in Pseudomyxoma peritonei: A short case series.

Pseudomyxoma peritonei (PMP) is a rare malignant disease characterized by a massive multifocal accumulation of mucin within the peritoneal cavity. The current treatment option is based on complete cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy. However, the recurrence is frequent with subsequent progression and death. To date, most of the studies published in PMP are related to histological and genomic analyses. Thus, the need for further studies unveiling the underlying PMP molecular mechanisms is urgent. In this regard, hypoxia and oxidative stress have been extensively related to tumoral pathologies, although their contribution to PMP has not been elucidated. In this manuscript, we have evaluated, for the first time, the intratumoral real-time oxygen microtension (pO2mt) in the tumor (soft and hard mucin) and surrounding healthy tissue from five PMP patients during surgery. In addition, we measured hypoxia (Hypoxia Inducible Factor-1a; HIF-1α) and oxidative stress (catalase; CAT) markers in soft and hard mucin from the same five PMP patient samples and in five control samples. The results showed low intratumoral oxygen levels, which were associated with increased HIF-1α protein levels, suggesting the presence of a hypoxic environment in these tumors. We also found a significant reduction in CAT activity levels in soft and hard mucin compared with healthy tissue samples. In conclusion, our study provides the first evidence of low intratumoral oxygen levels in PMP patients associated with hypoxia and oxidative stress markers. However, further investigation is required to understand the potential role of oxidative stress in PMP in order to find new therapeutic strategies.

Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway

ObjectivePhysical exercise, a common non-drug intervention, is an important strategy in cancer treatment, including hepatocellular carcinoma (HCC). However, the mechanism remains largely unknown. Due to the importance of hypoxia and cancer stemness in the development of HCC, the present study investigated whether the anti-HCC effect of physical exercise is related to its suppression on hypoxia and cancer stemness. MethodsA physical exercise intervention of swimming (30 min/d, 5 d/week, for 4 weeks) was administered to BALB/c nude mice bearing subcutaneous human HCC tumor. The anti-HCC effect of swimming was assessed in vivo by tumor weight monitoring, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) detection of proliferating cell nuclear antigen (PCNA) and Ki67. The expression of stemness transcription factors, including Nanog homeobox (NANOG), octamer-binding transcription factor 4 (OCT-4), v-Myc avian myelocytomatosis viral oncogene homolog (C-MYC) and hypoxia-inducible factor-1α (HIF-1α), was detected using real-time reverse transcription polymerase chain reaction. A hypoxia probe was used to explore the intratumoral hypoxia status. Western blot was used to detect the expression of HIF-1α and proteins related to protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway. The IHC analysis of platelet endothelial cell adhesion molecule-1 (CD31), and the immunofluorescence co-location of CD31 and desmin were used to analyze tumor blood perfusion. SMMC-7721 cells were treated with nude mice serum. The inhibition effect on cancer stemness in vitro was detected using suspension sphere experiments and the expression of stemness transcription factors. The hypoxia status was inferred by measuring the protein and mRNA levels of HIF-1α. Further, the expression of proteins related to Akt/GSK-3β/β-catenin signaling pathway was detected. ResultsSwimming significantly reduced the body weight and tumor weight in nude mice bearing HCC tumor. HE staining and IHC results showed a lower necrotic area ratio as well as fewer PCNA or Ki67 positive cells in mice receiving the swimming intervention. Swimming potently alleviated the intratumoral hypoxia, attenuated the cancer stemness, and inhibited the Akt/GSK-3β/β-catenin signaling pathway. Additionally, the desmin+/CD31+ ratio, rather than the number of CD31+ vessels, was significantly increased in swimming-treated mice. In vitro experiments showed that treating cells with the serum from the swimming intervention mice significantly reduced the formation of SMMC-7721 cell suspension sphere, as well as the mRNA expression level of stemness transcription factors. Consistent with the in vivo results, HIF-1α and Akt/GSK-3β/β-catenin signaling pathway were also inhibited in cells treated with serum from swimming group. ConclusionSwimming alleviated hypoxia and attenuated cancer stemness in HCC, through suppression of the Akt/GSK-3β/β-catenin signaling pathway. The alleviation of intratumoral hypoxia was related to the increase in blood perfusion in the tumor.Please cite this article as: Xiao CL, Zhong ZP, Lü C, Guo BJ, Chen JJ, Zhao T, Yin ZF, Li B. Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway. J Integr Med. 2023; 21(2): 184–193.

BAP1 is a novel regulator of HIF-1α

BAP1 is a powerful tumor suppressor gene characterized by haplo insufficiency. Individuals carrying germline BAP1 mutations often develop mesothelioma, an aggressive malignancy of the serosal layers covering the lungs, pericardium, and abdominal cavity. Intriguingly, mesotheliomas developing in carriers of germline BAP1 mutations are less aggressive, and these patients have significantly improved survival. We investigated the apparent paradox of a tumor suppressor gene that, when mutated, causes less aggressive mesotheliomas. We discovered that mesothelioma biopsies with biallelic BAP1 mutations showed loss of nuclear HIF-1α staining. We demonstrated that during hypoxia, BAP1 binds, deubiquitylates, and stabilizes HIF-1α, the master regulator of the hypoxia response and tumor cell invasion. Moreover, primary cells from individuals carrying germline BAP1 mutations and primary cells in which BAP1 was silenced using siRNA had reduced HIF-1α protein levels in hypoxia. Computational modeling and co-immunoprecipitation experiments revealed that mutations of BAP1 residues I675, F678, I679, and L691 -encompassing the C-terminal domain-nuclear localization signal- to A, abolished the interaction with HIF-1α. We found that BAP1 binds to the N-terminal region of HIF-1α, where HIF-1α binds DNA and dimerizes with HIF-1β forming the heterodimeric transactivating complex HIF. Our data identify BAP1 as a key positive regulator of HIF-1α in hypoxia. We propose that the significant reduction of HIF-1α activity in mesothelioma cells carrying biallelic BAP1 mutations, accompanied by the significant reduction of HIF-1α activity in hypoxic tissues containing germline BAP1 mutations, contributes to the reduced aggressiveness and improved survival of mesotheliomas developing in carriers of germline BAP1 mutations.

Murine herpesvirus-68-related growth factors treatment correlates with decrease of p53 and HIF-1α protein levels.

Murine herpesvirus 68 (MHV-68) belongs to the subfamily Gammaherpesvirinae of the family Herpesviridae. This exceptional murine herpesvirus is an excellent model for the study of human gammaherpesvirus infections. Cells infected with MHV-68 under nonpermissive conditions for viral replication produce substances designated as MHV-68 growth factors (MHGF-68), that can cause transformation of the cells, or on the other side, turn transformed cells into normal. It was already proposed, that the MHGF-68 fractions cause transformation, disruption of the cytoskeleton and slower growth of the tumors in nude mice. Here, we examined newly extracted fractions of MHGF-68 designated as F5 and F8. Both fractions proved to inhibit the growth of the spheroids and also tumours induced in nude mice. What more, the fractions caused the decrease of the protein levels of wt p53 and HIF-1α. Decreased levels of p53 and HIF-1α activity leads to decreased vascularization, slower tumour growth, and lower adaptation to hypoxic conditions. This would propose MHGF-68 fractions, or their human herpesvirus equivalents, as a potential anticancer drugs in combined chemotherapy.

Histopathological, ultrastructural, and immunohistochemical examination of changes in the placenta as a result of severe preeclampsia.

To investigate the role of hypoxia-inducible transcription factor-1 alpha (HIF-1α) and angiogenetic factor endothelin-1 (ET-1) expression in regulating hypoxia and placental development by routine histopathological methods. Twenty preeclamptic and normal placentas were used. Placenta tissue pieces were examined histopathologically after routine paraffin follow-ups. HIF-1α and ET-1 proteins were examined immunohistochemically, and placental tissues were examined ultrastructurally. Increase in syncytial proliferation, endothelial damage in vessels, and increase in collagen were observed in preeclamptic placentas. As a result of preeclampsia, an increase was observed in HIF-1α and ET-1 protein levels in the placenta. Dilatation of endoplasmic reticulum and loss of cristae in mitochondria were observed in trophoblast cells in preeclamptic placental sections. High regulation of oxygen resulting from preeclampsia has been shown to be a critical determinant of placentagenesis and plays an important role in placental differentiation, changes in maternal and fetal blood circulation, trophoblastic invasion, and syncytial node increase. It has been thought that preeclampsia affects secretion by disrupting the endoplasmic reticulum structure and induces mitochondrial damage, and that ET-1 may potentially help in the induction of stress pathways as a result of hypoxia in preeclampsia.