UNASSIGNED: Chimeric Antigen Receptor (CAR) T cell therapy has demonstrated remarkable success in treating hematological malignancies. However, its efficacy against solid tumors, including cervical cancer, remains a challenge. Hypoxia, a common feature of the tumor microenvironment, profoundly impacts CAR T cell function, emphasizing the need to explore strategies targeting hypoxia-inducible factor-1α (HIF-1α).
UNASSIGNED: In this study, we evaluated the effects of the HIF-1α inhibitor PX-478 on mesoCAR T cell function through in-silico and in vitro experiments. We conducted comprehensive analyses of HIF-1α expression in cervical cancer patients and examined the impact of PX-478 on T cell proliferation, cytokine production, cytotoxicity, and exhaustion markers.
UNASSIGNED: Our in-silico analyses revealed high expression of HIF-1α in cervical cancer patients, correlating with poor prognosis. PX-478 effectively reduced HIF-1α levels in T and HeLa cells. While PX-478 exhibited dose-dependent inhibition of antigen-nonspecific T and mesoCAR T cell proliferation, it had minimal impact on antigen-specific mesoCAR T cell proliferation. Notably, PX-478 significantly impaired the cytotoxic function of mesoCAR T cells and induced terminally exhausted T cells.
UNASSIGNED: Our results underscore the significant potential and physiological relevance of the HIF-1α pathway in determining the fate and function of both T and CAR T cells. However, we recognize the imperative for further molecular investigations aimed at unraveling the intricate downstream targets associated with HIF-1α and its influence on antitumor immunity, particularly within the context of hypoxic tumors. These insights serve as a foundation for the careful development of combination therapies tailored to counter immunosuppressive pathways within hypoxic environments and fine-tune CAR T cell performance in the intricate tumor microenvironment.

OBJECTIVE: To elucidate the key regulator responsible for autophagy and ferroptosis, and if specific pharmacological inhibitor of upregulated gene exerted the pro-autophagic and anti-ferroptotic effect on macrophage to alleviate the atherosclerosis.
METHODS: Autophagy and ferroptosis were evaluated in atherosclerotic lesions and THP-1 macrophages exposed to ox-LDL. Autophagy/ferroptosis-related differentially expressed genes (DEGs) in atherosclerosis were identified by bioinformatic analysis of GSE97210 dataset, and were validated in atherosclerotic cells and tissues. The efficacy and mechanism of pharmacological inhibition of the validated DEGs on alleviating atherosclerosis were explored in vivo and in vitro.
RESULTS: Atherosclerotic lesions were characterized by autophagy inhibition and ferroptosis activation in macrophages. The crosslink between autophagy and ferroptosis were demonstrated. Ox-LDL induced THP-1 macrophage foam cell formation, autophagy dysfunction, and ferroptosis occurrence. Rapamycin ameliorated and, conversely, erastin deteriorated the effect of ox-LDL on THP-1 macrophages. Eleven autophagy/ferroptosis-related DEGs were identified in atherosclerosis vs. normal. The up-regulated expression of HIF-1α was verified in atherosclerotic lesions and THP-1 macrophages induced by ox-LDL. HIF-1α inhibitor PX-478 restored autophagy function, depressed ferroptosis, and reduced lipid accumulation in ox-LDL induced THP-1 macrophage. Autophagy inhibitor 3-MA obviously abrogated the pro-autophagic, anti-ferroptotic, and anti-atherosclerotic effects of PX-478. PX-478 treatment down-regulated HIF-1α expression and reduced atherosclerotic plaques in the mice model.
CONCLUSIONS: Autophagy is inhibited, ferroptosis is activated, and crosslink occurs between autophagy and ferroptosis during atherosclerosis. HIF-1α, an upregulated DEG between atherosclerosis and normal, co-regulates autophagy and ferroptosis. HIF-1α inhibitor PX-478 attenuates foam cell formation and lessens atherosclerosis by enhancing autophagy and depressing ferroptosis in macrophages.

HIF1A is significantly upregulated in calcified human aortic valves (AVs). Furthermore, HIF1A inhibitor PX-478 was shown to inhibit AV calcification under static and disturbed flow conditions. Since elevated stretch is one of the major mechanical stimuli for AV calcification, we investigated the effect of PX-478 on AV calcification and collagen turnover under a pathophysiological cyclic stretch (15%) condition. Porcine aortic valve (PAV) leaflets were cyclically (1 Hz) stretched at 15% for 24 days in osteogenic medium with or without PX-478. In addition, PAV leaflets were cyclically stretched at a physiological (10%) and 15% for 3 days in regular medium to assess its effect of on HIF1A mRNA expression. It was found that 100 μM (high concentration) PX-478 could significantly inhibit PAV calcification under 15% stretch, whereas 50 μM (moderate concentration) PX-478 showed a modest inhibitory effect on PAV calcification. Nonetheless, 50 μM PX-478 significantly reduced PAV collagen turnover under 15% stretch. Surprisingly, it was observed that cyclic stretch (15% vs. 10%) did not have any significant effect on HIF1A mRNA expression in PAV leaflets. These results suggest that HIF1A inhibitor PX-478 may impart its anti-calcific and anti-matrix remodeling effect in a stretch-independent manner.

Atherosclerosis is a chronic multifactorial cardiovascular disease. Western diets have been reported to affect atherosclerosis through regulating adipose function. In high cholesterol diet-fed ApoE -/- mice, adipocyte HIF-1α deficiency or direct inhibition of HIF-1α by the selective pharmacological HIF-1α inhibitor PX-478 alleviates high cholesterol diet-induced atherosclerosis by reducing adipose ceramide generation, which lowers cholesterol levels and reduces inflammatory responses, resulting in improved dyslipidemia and atherogenesis. Smpd3, the gene encoding neutral sphingomyelinase, is identified as a new target gene directly regulated by HIF-1α that is involved in ceramide generation. Injection of lentivirus-SMPD3 in epididymal adipose tissue reverses the decrease in ceramides in adipocytes and eliminates the improvements on atherosclerosis in the adipocyte HIF-1α-deficient mice. Therefore, HIF-1α inhibition may constitute a novel approach to slow atherosclerotic progression.

To investigate the antitumor effects of the selective hypoxia-inducible factor-1 (HIF-1) inhibitors echinomycin and PX-478 on uterine fibroids.
Experimental study using in vitro primary culture systems and an in vivo mouse xenograft model.
Academic university center.
Women with uterine fibroids who underwent hysterectomy or myomectomy.
Administration of the selective HIF-1 inhibitors echinomycin and PX-478 to the media of the primary cultured uterine fibroid cells and to nonobese diabetic/severe combined immunodeficient mice bearing fibroid xenografts consisting of the primary cultured fibroid cells and type Ⅰ collagen gels beneath the kidney capsule.
Cell proliferation was measured by Cell Counting Kit-8 assay. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and by measuring caspase 3 and 7 activities. The xenografts were evaluated by gross appearance, surface area, and histology. The Ki-67 index was measured to evaluate proliferation of the xenografts.
Both echinomycin and PX-478 inhibited cell proliferation and induced apoptosis in fibroid cells cultured under hypoxia and normoxia. Enlargement of the fibroid xenografts was significantly attenuated. The Ki-67 index significantly decreased after the administration of the HIF-1 inhibitors in the xenograft model. Eight of 27 xenografts treated with the HIF-1 inhibitors contained calcification and hyalinizing components from 3 days after the grafting to 2 weeks, suggesting that the HIF-inhibitors induce degeneration of the fibroid xenografts.
The selective HIF-1 inhibitors echinomycin and PX-478 show antitumor effects against uterine fibroids both in vitro and in vivo. These findings support the potential use of HIF-1 inhibitors for the treatment of uterine fibroids.

Hypoxia inducible factor 1α (HIF1α) plays a critical role in atherosclerosis as demonstrated in endothelial-targeted HIF1α -deficient mice. However, it has not been shown if specific pharmacological inhibitors of HIF1α can be used as potential drugs for atherosclerosis. PX-478 is a selective inhibitor of HIF1α, which was used to reduce cancer and obesity in animal models. Here, we tested whether PX-478 can be used to inhibit atherosclerosis.
We first tested PX-478 in human aortic endothelial cells (HAEC) and found that it significantly inhibited expression of HIF1α and its targets, including Collagen I. Next, two independent atherosclerosis models, C57BL/6 mice treated with AAV-PCSK9 and ApoE-/- mice, were used to test the efficacy of PX-478. Both mouse models were fed a Western diet for 3 months with bi-weekly treatment with PX-478 (40 mg/kg) or saline.
PX-478 treatment reduced atherosclerotic plaque burden in the aortic trees in both mouse models, while plaque burden in the aortic sinus was reduced in the AAV-PCSK9 mouse model, but not in the ApoE-/- mice. Russell-Movat\'s Pentachrome and Picrosirius Red staining showed a significant reduction in extracellular matrix remodeling and collagen maturation, respectively, in the PX-478-treated mice. As expected, PX-478 treatment reduced diet-induced weight-gain and abdominal adipocyte hypertrophy. Interestingly, PX-478 reduced plasma LDL cholesterol by 69% and 30% in AAV-PCSK9 and ApoE-/- mice, respectively. To explore the cholesterol-lowering mechanisms, we carried out an RNA sequencing study using the liver tissues from the ApoE-/- mouse study. We found 450 genes upregulated and 381 genes downregulated by PX-478 treatment in the liver. Further, gene ontology analysis showed that PX-478 treatment upregulated fatty acid and lipid catabolic pathways, while downregulating lipid biosynthesis and plasma lipoprotein particle remodeling processes. Of interest, Cfd, Elovl3, and Insig2 were some of the most downregulated genes by PX-478, and have been implicated in fat storage, fatty acid elongation, and cholesterol metabolism. The downregulation of Cfd, Elovl3, and Insig2 was further validated by qPCR in the liver tissues of ApoE-/- mice treated with PX-478.
These results suggest that PX-478 is a potential anti-atherogenic drug, which targets vascular endothelium and hepatic cholesterol pathways.

BACKGROUND: One key approach for anticancer therapy is drug combination. Drug combinations can help reduce doses and thereby decrease side effects. Furthermore, the likelihood of drug resistance is reduced. Distinct alterations in tumor metabolism have been described in past decades, but metabolism has yet to be targeted in clinical cancer therapy. Recently, we found evidence for synergism between dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, and the HIF-1α inhibitor PX-478. In this study, we aimed to analyse this synergism in cell lines of different cancer types and to identify the underlying biochemical mechanisms.
METHODS: The dose-dependent antiproliferative effects of the single drugs and their combination were assessed using SRB assays. FACS, Western blot and HPLC analyses were performed to investigate changes in reactive oxygen species levels, apoptosis and the cell cycle. Additionally, real-time metabolic analyses (Seahorse) were performed with DCA-treated MCF-7 cells.
RESULTS: The combination of DCA and PX-478 produced synergistic effects in all eight cancer cell lines tested, including colorectal, lung, breast, cervical, liver and brain cancer. Reactive oxygen species generation and apoptosis played important roles in this synergism. Furthermore, cell proliferation was inhibited by the combination treatment.
CONCLUSIONS: Here, we found that these tumor metabolism-targeting compounds exhibited a potent synergism across all tested cancer cell lines. Thus, we highly recommend the combination of these two compounds for progression to in vivo translational and clinical trials.

Incomplete recovery from episodes of acute kidney injury (AKI) can predispose patients to develop chronic kidney disease (CKD). Although hypoxia-inducible factor-1α (HIF-1α) is a master regulator of the response to hypoxia/ischemia, the role of HIF-1α in CKD progression following incomplete recovery from AKI is poorly understood. Here, we investigated this issue using moderate and severe ischemia/reperfusion injury (I/RI) mouse models. We found that the outcomes of AKI were highly associated with the time course of tubular HIF-1α expression. Sustained activation of HIF-1α, accompanied by the development of renal fibrotic lesions, was found in kidneys with severe AKI. The AKI to CKD progression was markedly ameliorated when PX-478 (a specific HIF-1α inhibitor, 5 mg· kg-1·d-1, i.p.) was administered starting on day 5 after severe I/RI for 10 consecutive days. Furthermore, we demonstrated that HIF-1α C-terminal transcriptional activation domain (C-TAD) transcriptionally stimulated KLF5, which promoted progression of CKD following severe AKI. The effect of HIF-1α C-TAD activation on promoting AKI to CKD progression was also confirmed in in vivo and in vitro studies. Moreover, we revealed that activation of HIF-1α C-TAD resulted in the loss of FIH-1, which was the key factor governing HIF-1α-driven AKI to CKD progression. Overexpression of FIH-1 inhibited HIF-1α C-TAD and prevented AKI to CKD progression. Thus, FIH-1-modulated HIF-1α C-TAD activation was the key mechanism of AKI to CKD progression by transcriptionally regulating KLF5 pathway. Our results provide new insights into the role of HIF-1α in AKI to CKD progression and also the potential therapeutic strategy for the prevention of renal diseases progression.

BACKGROUND: Despite an improvement of prognosis in breast and colon cancer, the outcome of the metastatic disease is still severe. Microevolution of cancer cells often leads to drug resistance and tumor-recurrence. To target the driving forces of the tumor microevolution, we focused on synergistic drug combinations of selected compounds. The aim is to prevent the tumor from evolving in order to stabilize disease remission. To identify synergisms in a high number of compounds, we propose here a three-step concept that is cost efficient, independent of high-throughput machines and reliable in its predictions.
METHODS: We created dose response curves using MTT- and SRB-assays with 14 different compounds in MCF-7, HT-29 and MDA-MB-231 cells. In order to efficiently screen for synergies, we developed a screening tool in which 14 drugs were combined (91 combinations) in MCF-7 and HT-29 using EC25 or less. The most promising combinations were verified by the method of Chou and Talalay.
RESULTS: All 14 compounds exhibit antitumor effects on each of the three cell lines. The screening tool resulted in 19 potential synergisms detected in HT-29 (20.9%) and 27 in MCF-7 (29.7%). Seven of the top combinations were further verified over the whole dose response curve, and for five combinations a significant synergy could be confirmed. The combination Nutlin-3 (inhibition of MDM2) and PX-478 (inhibition of HIF-1α) could be confirmed for all three cell lines. The same accounts for the combination of Dichloroacetate (PDH activation) and NHI-2 (LDH-A inhibition). Our screening method proved to be an efficient tool that is reliable in its projections.
CONCLUSIONS: The presented three-step concept proved to be cost- and time-efficient with respect to the resulting data. The newly found combinations show promising results in MCF-7, HT-29 and MDA-MB231 cancer cells.

OBJECTIVE: Retinopathy of prematurity (ROP) is an important risk factor for blindness in children due to neovascularization (NV). Hypoxia stimulates the formation of NV, as retinal hypoxia affects the stability and function of hypoxia-inducible factor (HIF) transcription factors. The purpose of this study is to study the mechanism of ROP and provide theoretical basis for clinical treatment of ROP.
OBJECTIVE: In the present study, we used a mouse model of oxygen-induced retinopathy (OIR) to demonstrate the effects of the HIF-1α inhibitor PX-478 on OIR, and to determine its mechanism of action, to provide a theoretical basis for the clinical treatment of ROP.
METHODS: The OIR mouse model was induced by exposing neonatal mouse pups and their mothers to 75 ± 5% oxygen from postnatal day 7 (P7) to P12, before being returned to room air from P12 to P17. Flat mount analyses were performed at P12 and P17. Hif1a, Hif2a, Hif3a, and Vegfa mRNA were detected by reverse transcription-polymerase chain reaction in OIR mice at P12 and P17. Hif1a and Vegfa mRNA were detected in OIR mice at P12 and P17 treatment with PX-478. Western blot analyses were used to assess the levels of HIF-1α, VEGF-A, and EPO before and after treatment with PX-478 at P12 and P17.
RESULTS: Hif1a mRNA was increased in OIR mice at P12 and P17, while Vegfa mRNA was increased at P12 and P17. HIF-1α, VEGF-A, and EPO protein levels were increased in OIR mice at P12 and P17, as compared to control mice at the same age (all p < 0.05). Inhibition of HIF-1α by injection of PX-478 in OIR mice (P9-P16) caused a decrease in the retinal avascular area at P12 and P17 (both p < 0.05), NV areas at P17 (p < 0.05), Vegfa mRNA decreased at P12 and P17, as compared to control mice (p < 0.05), and VEGF-A and EPO protein levels at P12 and P17, as compared to control mice. Our study found that there were PX-478 both retina and vitreous body of OIR. Inhibition of HIF-1α by injection of PX-478 in OIR mice caused a decrease in the retinal avascular area at P12 and P17, NV areas decreased at P17, VEGF-A and EPO protein levels at P12 and P17. Endothelial cell migration assays and cell tube formation indication PX-478 attenuate cell migration and significantly weakened the cell cavity formation under the condition of hypoxia.
CONCLUSIONS: HIF-1α plays a main role in OIR and can be considered a therapeutic target in OIR by suppressing downstream angiogenic factors, PX-478 decreasing the retinal avascular area and NV.