FIH-1-modulated HIF-1α C-TAD promotes acute kidney injury to chronic kidney disease progression via regulating KLF5 signaling.

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.

Synergisms of genome and metabolism stabilizing antitumor therapy (GMSAT) in human breast and colon cancer cell lines: a novel approach to screen for synergism.

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.

Inhibition of HIF-1α enhances anti-tumor effects of dendritic cell-based vaccination in a mouse model of breast cancer.

Considerable evidence shows that the tumor microenvironment is an active participant in preventing immunosurveillance and limiting the efficacy of anticancer therapies. Hypoxia is a prominent characteristic of the solid tumor microenvironment. The transcription factor hypoxia-inducible factor-1α (HIF-1α) is an important mediator of hypoxic response of tumor cells that modulates the expression of specific genes involved in tumor immunosuppression. Using a 4T1 breast cancer model, we show that in vivo administration of PX-478, an inhibitor of oxygen-sensitive HIF-1α, led to reduced expression of Foxp3 and VEGF transcript and/or protein, molecules that are directly controlled by HIF-1. When combined with dendritic cell (DC)-based vaccination, HIF-1α inhibition resulted in an augmented cytotoxic T lymphocyte effector function, improved proliferation status of T cells, increased production of inflammatory cytokine IFN-γ, as well as reduced regulatory function of T cells in association with slower tumor growth. Taken together, our findings indicate that the use of HIF-1α inhibition provides an immune adjuvant activity, thereby improves the efficacy of tumor antigen-based DC vaccine.

HIF-1alpha and cancer therapy.

Most solid tumors develop regions of hypoxia as they grow and outstrip their blood supply. In order to survive in the stressful hypoxic environment, tumor cells have developed a coordinated set of responses orchestrating their adaptation to hypoxia. The outcomes of the cellular responses to hypoxia are aggressive disease, resistance to therapy, and decreased patient survival. A critical mediator of the hypoxic response is the transcription factor hypoxia-inducible factor 1 (HIF-1) that upregulates expression of proteins that promote angiogenesis, anaerobic metabolism, and many other survival pathways. Regulation of HIF-1alpha, a component of the HIF-1 heterodimer, occurs at multiple levels including translation, degradation, and transcriptional activation, and serves as a testimony to the central role of HIF-1. Studies demonstrating the importance of HIF-1alpha expression for tumor survival have made HIF-1alpha an attractive target for cancer therapy. The growing l.ist of pharmacological inhibitors of HIF-1 and their varied targets mirrors the complex molecular mechanisms controlling HIF-1. In this chapter, we summarize recent findings regarding the regulation of HIF-1alpha and the progress made in identifying new therapeutic agents that inhibit HIF-1alpha.

Vasculogenic mimicry formation in EBV-associated epithelial malignancies.

Epstein-Barr virus (EBV)-associated epithelial cancers, including nasopharyngeal carcinoma (NPC) and approximately 10% of gastric cancers, termed EBVaGC, represent 80% of all EBV-related malignancies. However, the exact role of EBV in epithelial cancers remains elusive. Here, we report that EBV functions in vasculogenic mimicry (VM). Epithelial cancer cells infected with EBV develop tumor vascular networks that correlate with tumor growth, which is different from endothelial-derived angiogenic vessels and is VEGF-independent. Mechanistically, activation of the PI3K/AKT/mTOR/HIF-1α signaling cascade, which is partly mediated by LMP2A, is responsible for EBV-induced VM formation. Both xenografts and clinical samples of NPC and EBVaGC exhibit VM histologically, which are correlated with AKT and HIF-1α activation. Furthermore, although anti-VEGF monotherapy shows limited effects, potent synergistic antitumor activities are achieved by combination therapy with VEGF and HIF-1α-targeted agents. Our findings suggest that EBV creates plasticity in epithelial cells to express endothelial phenotype and provides a novel EBV-targeted antitumor strategy.

Comparative pharmacokinetics of 1-(2-chloroethyl)-3-(2,6-dioxo-1-piperidyl)-1-nitrosourea in rats and patients and extrapolation to clinical trials.

The plasma pharmacokinetics of 1-(2-chloroethyl)-3-(2,6-dioxo-1-piperidyl)1-nitrosourea (PCNU) were determined in ambulatory rats and in patients receiving PCNU chemotherapy in Phase 1 and II studies. After derivativization to the methyl carbamate, both rat and human PCNU plasma levels were measured by gas chromatography-mass spectrometry. Comparison of the tolerated dose levels and pharmacokinetics of PCNU to the values determined for 1,3-bis(2-chloroethyl)-1-nitrosourea in humans indicated that PCNU has a lower plasma drug area under the curve at equitoxic doses. We conclude that PCNU may show less clinical efficacy than 1,3-bis(2-chloroethyl)-1-nitrosourea in the treatment of solid tumors.

Small molecule-mediated anti-cancer therapy via hypoxia-inducible factor-1 blockade.

Despite reservations regarding potential toxicities, small molecule-mediated blockade of the hypoxia-inducible factor-1 transcription factor has emerged as a viable anti-cancer strategy in vivo. Recent experiments by Welsh et al. revealed unprecedented anti-tumor responses of various aggressive solid tumors to the HIF-1-inhibitory small molecule drug PX-478. Compared with other anti-cancer drugs, PX-478 had markedly improved regression, growth delay and log10 cell kill profiles, particularly against large tumors that are normally refractory to small molecule drug therapy. Importantly, pharmacokinetic and toxicity profiles were within acceptable limits, providing rationale for the clinical development of HIF-1 inhibitors in general. Though the mechanism of action for PX-478 is not completely understood, inhibition of glycolysis rather than angiogenesis appeared to be the primary mode of anti-cancer activity.

Chemoimmunotherapy of cancer.

The preparation of a series of water-soluble mustard haptens for chemoimmunotherapy of cancer is described. Preliminary screening data are given, indicating some activity against P388 lymphocytic leukemia for those compounds containing the most potent immunogenic functional groups.

Abnormal spleen scan following MOPP therapy in a patient with Hodgkin's disease: case report.

A patient with Stage IV-A nodular sclerosing Hodgkin's disease is described who, at initial presentation, demonstrated an apparent large filling defect in the spleen by 99mTc-sulfur colloid scan. Following a 6-month course of MOPP therapy, the patient was clinically free of disease although the spleen scan remained unchanged. After laparotomy, the spleen was found to be entirely normal histologically.

Phase II trail of PCNU in refractory advanced breast cancer.

An evaluation of PCNU was carried out in 28 patients with extensively treated refractory breast cancer. The starting dose was 60 mg/m2 in 13 patients and 90 mg/m2 in 15 patients given intravenously every 6 weeks. The major side effect was myelosuppression, manifested mainly as thrombocytopenia. Nonhematologic side effects were minimal, consisting mainly of transient nausea. One mixed response was seen. Four patients had stable disease. PCNU demonstrated limited activity in advanced breast cancer and was not effective in the treatment of central nervous system metastases.

Substances with antineoplastic activity. XCVI. Some pharmacological properties and therapeutic synergism of 6-purinyl-N-(2-chloroethyl)thiocarbamate with cytosine arabinoside.

The cancerostatic effect of 6-purinyl-N-(2-chloroethyl)thiocarbamate (Cloturin VUFB, VUFB-15686) was studied in detail in mice and rats bearing transplantable tumors. The cytotoxic activities were measured by determining the incorporation rate of 5-iodo-2'-deoxy[6-3H]uridine and uniformly labeled [U-14C]amino acid mixture into trichloroacetic acid-insoluble fraction of Yoshida ascites reticulosarcoma cells during a short-term incubation with the drug. From the results obtained it follows that the new substance is therapeutically more effective in comparison with 6-mercaptopurine (NSC-755). A therapeutic synergism of Cloturin with cytosine arabinoside (NSC-63978) was observed.

Chronic hyperpigmentation from a heated mustard compress burn: a case report.

This is a case study of a woman who sustained a second-degree, partial-thickness burn of the midchest and medial breast distribution after the application of a heated mustard compress as a naturopathic remedy for a recent bout of pneumonia. This case study demonstrates the potential for hyperpigmentation and hypertrophic scarring associated with heated mustard burns. A photograph of the presenting burn with the region of hyperpigmentation 3 days after the injury is provided.

Selective inhibition of hypoxia-inducible factor 1α ameliorates adipose tissue dysfunction.

Hypoxia-inducible factor 1α (HIF1α) induction in adipocytes is a critical component of the "fibrotic response," directly linked to metabolic dysfunction in adipose tissues under hypoxic conditions. We reasoned that inhibition of HIF1α may ameliorate the negative aspects of the obesity-associated fat pad expansion. We used the selective HIF1α inhibitor PX-478, whose effectiveness has previously been established in tumor models. We demonstrate that PX-478 treatment effectively suppresses the high-fat-diet (HFD)-induced HIF1α activation in adipose tissue. HIF1α inhibition causes a reduction of weight gain in mice on an HFD but not on a chow diet. Treatment increases energy expenditure and prompts resistance to HFD-mediated deterioration of metabolic parameters. Moreover, PX-478-treated mice have reduced fibrosis and fewer inflammatory infiltrates in their adipose tissues. We confirm the metabolic effects obtained with PX-478 treatment using an adipose tissue-specific, doxycycline-inducible dominant negative HIF1α mutant (dn-HIF1α). Consistent with the pharmacological results, genetic inhibition of endogenous HIF1α activity prompts similar metabolic improvements in HFD-fed mice. Collectively, our results demonstrate that HIF1α inhibition in the adipocyte leads to significant metabolic improvements, suggesting that selective HIF1α inhibition in adipose tissue may be an effective therapeutic avenue in the context of metabolic dysfunction.

Hypoxia-inducible factor inhibitors: a survey of recent patented compounds (2004 - 2010).

INTRODUCTION: Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor consisting of α and ß subunits that regulates the expression of angiogenic factors, including VEGF, which are involved in angiogenesis, invasion/metastasis, glucose uptake and cell survival during cancer development. AREAS COVERED: This review summarizes the information about patented HIF inhibitors over the last 7 years (2004 - 2010). The reader will gain an outline of the structure and biological activity of recently developed HIF inhibitors. EXPERT OPINION: Inhibition of HIF is an attractive therapeutic target for tumor angiogenesis and, until now, various HIF inhibitors have been discovered and evaluated. It is expected that development of more potent and selective HIF inhibitors will provide an effective treatment of cancer and other HIF-related diseases, including inflammation and cardiovascular disorder. As VEGF plays an important role in angiogenesis during tumor growth and ischemic diseases, the inhibition of VEGF-induced HIF is an attractive approach for the suppression of hypoxia-mediated pathological angiogenesis. HIF inhibitors may not only have cytostatic antitumor effects with fewer side effects, but also synergetic effects combined with radiotherapy.

Treatment with HIF-1alpha antagonist PX-478 inhibits progression and spread of orthotopic human small cell lung cancer and lung adenocarcinoma in mice.

INTRODUCTION: PX-478 is a potent small-molecule inhibitor of hypoxia-inducible factor 1alpha (HIF-1alpha). In prior preclinical studies, it had antitumor activity against various solid tumors in subcutaneous xenografts but had no measurable activity against a non-small cell lung cancer (NSCLC) xenograft. To determine the effectiveness of PX-478 against lung tumors, we investigated HIF-1alpha expression in several lung cancer cell lines, both in vitro and in vivo, and treated orthotopic mouse models of human lung cancer with PX-478. METHODS: Cells from two human lung adenocarcinoma cell models (PC14-PE6 and NCI-H441) or two human small cell lung cancer (SCLC) models (NCI-H187 and NCI-N417) were injected into the left lungs of nude mice and were randomized 16 to 18 days after injection with daily oral treatment with PX-478 or vehicle for 5 days. RESULTS: In the PC14-PE6 NSCLC model, treatment with 20 mg/kg PX-478 significantly reduced the median primary lung tumor volume by 87% (p = 0.005) compared with the vehicle-treated group. PX-478 treatment also markedly reduced mediastinal metastasis and prolonged survival. Similar results were obtained in a second NSCLC model. In SCLC models, PX-478 was even more effective. In the NCI-H187 model, the median primary lung tumor volume was reduced by 99% (p = 0.0001). The median survival duration was increased by 132%. In the NCI-N417 model, the median primary lung tumor volume was reduced by 97% (p = 0.008). CONCLUSIONS: We demonstrated that the PX-478, HIF-1alpha inhibitor, had significant antitumor activity against two orthotopic models of lung adenocarcinomas and two models of SCLC. These results suggest the inclusion of lung cancer patients in phase I clinical trials of PX-478.