Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an inherited cancer syndrome linked to biallelic inactivation of the gene encoding the tricarboxylic acid cycle enzyme fumarate hydratase (FH).Individuals with HLRCC are at risk to develop cutaneous and uterine leiomyomas and an aggressive form of kidney cancer. Pseudohypoxic drive-the aberrant activation of cellular hypoxia response pathways despite normal oxygen tension-is considered to be a likely mechanism underlying the etiology of this tumor. Pseudohypoxia requires the oxygen-independent stabilization of the ␣ subunit of the hypoxia-inducible transcription factor (HIF-1␣). Under normoxic conditions, proline hydroxylation of HIF-1␣ permits VHL recognition and subsequent targeting for proteasomal degradation. Here, we demonstrate that inactivating mutations of FH in an HLRCC-derived cell line result in glucose-mediated generation of cellular reactive oxygen species (ROS) and ROS-dependent HIF-1␣ stabilization. Additionally, we demonstrate that stable knockdown of FH in immortalized renal epithelial cells results in ROS-dependent HIF-1␣ stabilization. These data reveal that the obligate glycolytic switch present in HLRCC is critical to HIF stabilization via ROS generation.Patients with hereditary leiomyomatosis and renal cell cancer (HLRCC) harbor germ line mutations of the FH gene, which encodes the tricarboxylic acid cycle enzyme fumarate hydratase, and affected individuals are at risk for the development of leiomyomas of the skin and uterus (fibroids) as well as kidney cancer (11,25,37). Genetic analysis of tumor samples indicates that FH acts as a tumor suppressor gene (37). The renal tumors that develop in HLRCC patients are notable for their aggressiveness, and effective systemic therapies are lacking at this time. Hence, identification of the molecular mechanisms that underlie the pathogenesis of this disease is needed to facilitate the development of targeted therapeutic strategies. Moreover, such studies may provide further insight into the role of mitochondrial metabolism in both normal and aberrant cellular physiology.FH catalyzes the enzymatic step of the tricarboxylic (TCA) cycle that hydrates fumarate to form malate. Proposed mechanisms for HLRCC tumor formation include apoptotic resistance, oxidative stress, and pseudohypoxic drive (10). Of these, most reports to date support a role for pseudohypoxic drive, based specifically on studies of hypoxia-inducible transcription factor 1␣ (HIF-1␣) expression. Pseudohypoxia is defined as the aberrant activation of hypoxia response pathways under normal oxygen conditions. HIF-1␣ expression is elevated both in HLRCC tumor specimens and in normoxic cells in which FH expression has been transiently suppressed with small interfering RNA (siRNA) (16). HIF-2␣ expression is also elevated in HLRCC tumor samples, although to a lesser extent than is HIF-1␣. In addition, there is clear evidence of upregulated transcription of HIF target genes in HLRCC tumor samples and in FH siRNA-treated cells (16,30). Fur...

Pioglitazone is used globally for the treatment of type 2 diabetes mellitus (T2DM) and is one of the most effective therapies for improving glucose homeostasis and insulin resistance in T2DM patients. However, its mechanism of action in the tissues and pathways that regulate glucose metabolism are incompletely defined. Here we investigated the direct effects of pioglitazone on hepatocellular pyruvate metabolism and the dependency of these observations on the purported regulators of mitochondrial pyruvate transport, MPC1 and MPC2. In cultured H4IIE hepatocytes, pioglitazone inhibited [2-14C]-pyruvate oxidation and pyruvate-driven oxygen consumption and, in mitochondria isolated from both hepatocytes and human skeletal muscle, pioglitazone selectively and dose-dependently inhibited pyruvate-driven ATP synthesis. Pioglitazone also suppressed hepatocellular glucose production (HGP), without influencing the mRNA expression of key HGP regulatory genes. Targeted siRNA silencing of MPC1 and 2 caused a modest inhibition of pyruvate oxidation and pyruvate-driven ATP synthesis, but did not alter pyruvate-driven HGP and, importantly, it did not influence the actions of pioglitazone on either pathway. In summary, these findings outline a novel mode of action of pioglitazone relevant to the pathogenesis of T2DM and suggest that targeting pyruvate metabolism may lead to the development of effective new T2DM therapies.

Radiotherapy is a common treatment for prostate cancer, but failure is observed 30 to 40% of the time. It is more common in patients with abnormal p53. The phytochemical diferuloylmethane (curcumin) a naturally occurring fl avinoid derived from the rhizome of Curcuma longa, shows potential radiosensitizing effects. In the present study, the effect of curcumin and radiation on cell viability, apoptosis and clonogenic cell death was examined in LNCaP (wild type p53) and PC3 (mutant p53) prostate cancer cells.The expression of p53 and p53 target genes was examined using RNase protection assay and Western blot analysis. Cell cycle and apoptosis was evaluated by fl ow cytometry. Cell viability and colony formation was examined using MTT assay and clonogenic assay respectively.The expression of p53, p21 and gadd45 increased in LNCaP cells after radiation exposure. In PC3 cells, there was no change in expression in mutant p53 after radiation or curcumin treatment. However, the expression of p21 and gadd45 increased after curcumin treatment in PC3 cells independent of p53. Curcumin induced cell growth arrest and apoptosis in both the cell lines. The cell viability and cell proliferation decreased in presence of both curcumin and radiation as compared to curcumin or radiation alone in both the cell lines. Regardless of p53 status, combination treatment with curcumin (2.5 to 10 μM) and radiation (2 Gy) had a synergistic effect on clonogenic cell death in both LNCaP and PC3 cells. Conclusion:Curcumin appears to radiosensitize prostate cancer cells and may be a possible adjuvant to radiotherapy in the treatment of prostate cancers.

The objective of this study was to investigate whether exposure of human monocytes to a pulsed ultra-wideband electromagnetic field (EMF) of 1 kV/cm average peak power triggers a signaling pathway responsible for the transcriptional regulation of NFKB (NF-kappaB)-dependent gene expression. Human Mono Mac 6 (MM6) cells were exposed intermittently to EMF pulses for a total of 90 min. The pulse width was 0.79+/-0.01 ns and the pulse repetition rate was 250 pps. The temperature of the medium was maintained at 37 degrees C in both sham- and EMF-exposed flasks. Total NFKB DNA-binding activity was measured in the nuclear extracts by the electrophoretic mobility shift assay. Cells exposed to the EMFs and incubated for 24 h postexposure showed a 3.5+/-0.2-fold increase in the NFKB DNA-binding activity. Since activation of NFKB was observed, the possibility of kappaB-dependent gene expression in response to exposure to the EMFs was investigated using NFKB signal-specific gene arrays. The results revealed no difference in the NFKB-dependent gene expression profiles at 8 or 24 h postexposure, indicating that activated NFKB does not lead to the differential expression of kappaB-dependent target genes. To determine whether the absence of the kappaB-dependent gene expression was due to compromised transcriptional regulation of NFKB, the functional activity of NFKB was examined in cells transiently transfected with Mercury Pathway constructs containing 4x NFKB binding sites associated either with the luciferase reporter system or a control vector. Pulsed EMF exposure did not induce NFKB-driven luciferase activity in these cells, indicating that the activation of NFKB at 24 h after the 1 kV/cm EMF exposure is functionally inactive. From these results, it is clear that the EMF-induced NFKB activation is only a transient response, with minimal or no downstream effect.

AimsFFA and FFA metabolites cause insulin resistance and impair beta cell function. The goal of our research was to examine whether elevation of plasma FFA impairs mitochondrial function and alters PGC-1α promoter methylation.MethodsIn this uncontrolled, change from baseline study design, insulin sensitivity and glucose-stimulated insulin secretion were measured in 9 normal glucose tolerant subjects before and after 3 day lipid infusion to elevate plasma FFA concentration. Vastus lateralis muscle biopsies were obtained and mitochondrial function, PGC-1α expression, and PGC-1α promoter methylation were quantitated.ResultsIncreased plasma FFA (440±93 μmol/Lto 997±242 μM, p<0.001) decreased insulin-stimulated total glucose disposal (TGD) by 25% (p = 0.008), impaired suppression of endogenous glucose production (p = 0.01), and reduced mitochondrial ATP synthesis with complex 1 (34%, p<0.05) and complex 2 (30%, p<0.05) substrates. Lipid infusion had no effect on muscle PGC-1α RNA expression, total methylation or non-CpG methylation, but methylation of the alternative PGC-1α promoter decreased (1.30±0.30 to 0.84±0.15% methylated residues/patient•strand, p = 0.055). Within PGC-1α promoter there was demethylation of CpT residues (0.72±0.16 vs. 0.28±0.10 methylated residues/patient•strand) (p = 0.002), which was inversely correlated with PGC-1α mRNA expression (r = -0.94, p<0.0001) and ATP synthesis with complex 1 (r = -0.80, p<0.01) and complex 2 (r = -0.69, p<0.05) substrates. Lipid infusion increased DNMT-3B (methyltransferase associated with PGC-1α promoter non-CpG methylation) mRNA expression (0.87 ± 0.09 to 1.62 ± 0.22 arbitrary units, p = 0.005), which correlated inversely with CpT demethylation (r = 0.67, p<0.05).Conclusion/InterpretationPhysiologic plasma FFA elevation in NGT individuals has opposing effects on PGC-1α non-CpG residue methylation (CpT demethylation and increased DNMT-3B expression), which is correlated with changes in PGC-1α expression and skeletal muscle mitochondrial function.