Huiyun Liang

The in vivo Gene Expression Signature of Oxidative Stress

How higher organisms respond to elevated oxidative stress in vivo is poorly understood. Therefore, we measured oxidative stress parameters and gene expression alterations (Affymetrix arrays) in the liver caused by elevated reactive oxygen species induced in vivo by diquat or by genetic ablation of the major antioxidant enzymes CuZn-superoxide dismutase (Sod1) and glutathione peroxidase-1 (Gpx1). Diquat (50 mg/kg) treatment resulted in a significant increase in oxidative damage within 3-6 h in wild-type mice without any lethality. In contrast, treatment of Sod1(-/-) or Gpx1(-/-) mice with a similar concentration of diquat resulted in a significant increase in oxidative damage within an hour of treatment and was lethal, i.e., these mice are extremely sensitive to the oxidative stress generated by diquat. The expression response to elevated oxidative stress in vivo does not involve an upregulation of classic antioxidant genes, although long-term oxidative stress in Sod1(-/-) mice leads to a significant upregulation of thiol antioxidants (e.g., Mt1, Srxn1, Gclc, Txnrd1), which appears to be mediated by the redox-sensitive transcription factor Nrf2. The main finding of our study is that the common response to elevated oxidative stress with diquat treatment in wild-type, Gpx1(-/-), and Sod1(-/-) mice and in untreated Sod1(-/-) mice is an upregulation of p53 target genes (p21, Gdf15, Plk3, Atf3, Trp53inp1, Ddit4, Gadd45a, Btg2, Ndrg1). A retrospective comparison with previous studies shows that induction of these p53 target genes is a conserved expression response to oxidative stress, in vivo and in vitro, in different species and different cells/organs.

Whole body overexpression of PGC-1α has opposite effects on hepatic and muscle insulin sensitivity

Type 2 diabetes is characterized by fasting hyperglycemia, secondary to hepatic insulin resistance and increased glucose production. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) is a transcriptional coactivator that is thought to control adaptive responses to physiological stimuli. In liver, PGC-1alpha expression is induced by fasting, and this effect promotes gluconeogenesis. To examine whether PGC-1alpha is involved in the pathogenesis of hepatic insulin resistance, we generated transgenic (TG) mice with whole body overexpression of human PGC-1alpha and evaluated glucose homeostasis with a euglycemic-hyperinsulinemic clamp. PGC-1alpha was moderately (approximately 2-fold) overexpressed in liver, skeletal muscle, brain, and heart of TG mice. In liver, PGC-1alpha overexpression resulted in increased expression of hepatocyte nuclear factor-4alpha and the gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. PGC-1alpha overexpression caused hepatic insulin resistance, manifested by higher glucose production and diminished insulin suppression of gluconeogenesis. Paradoxically, PGC-1alpha overexpression improved muscle insulin sensitivity, as evidenced by elevated insulin-stimulated Akt phosphorylation and peripheral glucose disposal. Content of myoglobin and troponin I slow protein was increased in muscle of TG mice, indicating fiber-type switching. PGC-1alpha overexpression also led to lower reactive oxygen species production by mitochondria and reduced IKK/IkappaB signaling in muscle. Feeding a high-fat diet to TG mice eliminated the increased muscle insulin sensitivity. The dichotomous effect of PGC-1alpha overexpression in liver and muscle suggests that PGC-1alpha is a fuel gauge that couples energy demands (muscle) with the corresponding fuel supply (liver). Thus, under conditions of physiological stress (i.e., prolonged fast and exercise training), increased hepatic glucose production may help sustain glucose utilization in peripheral tissues.

PGC-1α protects neurons and alters disease progression in an amyotrophic lateral sclerosis mouse model

Introduction: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. We sought to determine whether peroxisome proliferator–activated receptor γ coactivator 1α (PGC‐1α) would have a beneficial effect on this disease. Methods: PGC‐1α transgenic mice were crossed with SOD1 mutant G93A DL mice. Results: We observed a moderate but non‐significant increase in average lifespan in PGC‐1α/G93A DL mice, as compared with G93A DL mice (292 ± 3 days vs. 274 ± 7 days). Although the onset of ALS was not altered, progression of the disease was significantly slower (∽34% increase in duration) in the PGC‐1α/G93A DL mice. These mice also exhibited markedly improved performance on the rotarod test, and the improved motor activity was associated with a decreased loss of motor neurons and less degeneration of neuromuscular junctions. Conclusion: A sustained level of excitatory amino acid transporter protein 2 (EAAT2) in astrocytes of the PGC‐1α/G93A DL mice may contribute to neuronal protection. Muscle Nerve 2011

Grb10 Promotes Lipolysis and Thermogenesis by Phosphorylation-dependent Feedback Inhibition of mTORC1

Identification of key regulators of lipid metabolism and thermogenic functions has important therapeutic implications for the current obesity and diabetes epidemic. Here, we show that Grb10, a direct substrate of mechanistic/mammalian target of rapamycin (mTOR), is expressed highly in brown adipose tissue, and its expression in white adipose tissue is markedly induced by cold exposure. In adipocytes, mTOR-mediated phosphorylation at Ser501/503 switches the binding preference of Grb10 from the insulin receptor to raptor, leading to the dissociation of raptor from mTOR and downregulation of mTOR complex 1 (mTORC1) signaling. Fat-specific disruption of Grb10 increased mTORC1 signaling in adipose tissues, suppressed lipolysis, and reduced thermogenic function. The effects of Grb10 deficiency on lipolysis and thermogenesis were diminished by rapamycin administration in vivo. Our study has uncovered a unique feedback mechanism regulating mTORC1 signaling in adipose tissues and identified Grb10 as a key regulator of adiposity, thermogenesis, and energy expenditure.

Apoptosis induction by the dual-action DNA- and protein-reactive antitumor drug irofulven is largely Bcl-2-independent

The overexpression of Bcl-2 is implicated in the resistance of cancer cells to apoptosis. This study explored the potential of irofulven (hydroxymethylacylfulvene, HMAF, MGI 114, NSC 683863), a novel DNA- and protein-reactive anticancer drug, to overcome the anti-apoptotic properties of Bcl-2 in HeLa cells with controlled Bcl-2 overexpression. Irofulven treatment resulted in rapid (12hr) dissipation of the mitochondrial membrane potential, phosphatidylserine externalization, and apoptotic DNA fragmentation, with progressive changes after 24hr. Bcl-2 overexpression caused marginal or partial inhibition of these effects after treatment times ranging from 12 to 48hr. Both Bcl-2-dependent and -independent responses to irofulven were abrogated by a broad-spectrum caspase inhibitor. Despite the somewhat decreased apoptotic indices, cell growth inhibition by irofulven was unaffected by Bcl-2 status. In comparison, Bcl-2 overexpression drastically reduced apoptotic DNA fragmentation by etoposide, acting via topoisomerase II-mediated DNA damage, but had no effect on apoptotic DNA fragmentation by helenalin A, which reacts with proteins but not DNA. Irofulven retains its pro-apoptotic and growth inhibitory potential in cell lines that have naturally high Bcl-2 expression. Collectively, the results implicate multiple mechanisms of apoptosis induction by irofulven, which may differ in time course and Bcl-2 dependence. It is possible that the sustained ability of irofulven to induce profound apoptosis and to block cell growth despite Bcl-2 overexpression may be related to its dual reactivity with both DNA and proteins.

Irofulven Induces Apoptosis in Breast Cancer Cells Regardless of Caspase-3 Status*

Caspase-3 deficiency can limit the efficiency of pro-apoptotic anticancer treatments. Irofulven (hydroxymethylacylfulvene, HMAF, MGI 114, NSC 683863) is an antitumor drug, currently in a Phase III and multiple Phase II trials, which can differentiate between tumor and normal cells in apoptosis induction. This study investigated whether apoptosis induced by irofulven requires caspase-3. Irofulven action was compared in breast cancer cells differing in caspase-3 status: deficient MCF-7 cells and proficient MDA-MB-231 cells and in normal human mammary epithelial cells, HMEC. Irofulven induces significant, concentration and time-dependent apoptotic DNA fragmentation in breast cancer cell lines, regardless of caspase-3 status. After 12, 24 and 48 h incubation at 1 μM irofulven (∼ 3 × GI50), fragmented DNA comprised 3.7, 14.1 and 34.6% and 8.4, 12.6 and 20.3% of total DNA in MCF-7 and MDA-MB-231 cells, respectively. Cell viability (trypan blue exclusion) remained largely unaffected during the first 24 h but decreased markedly after 48 h, indicating secondary necrosis. Net losses in cell numbers were apparent at 48 h. Normal HMEC cells were refractory to 1 μM drug with only ∼3–9% fragmented DNA after 12–48 h, although apoptosis was observed at drug levels >3 μM. The broad-spectrum caspase inhibitor Z-VAD-fmk inhibited irofulven-induced apoptosis of all cell lines at 20 μM with nearly complete abrogation of apoptosis at 100 μM. Irofulven treatment resulted in marginal caspase-3 processing in MDA-MB-231 and HMEC cells. These results indicate that whereas the caspase cascade mediates irofulven- induced apoptosis, caspase-3 is dispensable (supported by NIH CA70091 and CA78706).

Resveratrol and P-glycoprotein Inhibitors Enhance the Anti-Skin Cancer Effects of Ursolic Acid

Ursolic acid, present in apples, rosemary, and other sources, is known to inhibit tumor formation and tumor cell viability in multiple systems, including skin. However, various cancers are resistant to ursolic acid treatment. Herein, skin carcinoma cells (Ca3/7) as compared with skin papilloma cells (MT1/2) displayed more resistance to ursolic acid-induced cytotoxicity. Interestingly, Ca3/7 cells had elevated levels of P-glycoprotein (P-gp), an ATP-dependent efflux pump that mediates resistance to chemotherapy in preclinical and clinical settings, and not only accumulated less but also more rapidly expelled the P-gp substrate rhodamine 123 (Rh123) indicating ursolic acid is transported by P-gp. To determine whether P-gp inhibition can enhance ursolic acid-mediated cytotoxicity, cells were challenged with P-gp inhibitors verapamil or cyclosporin A. Alternatively, cells were pretreated with the natural compound resveratrol, a known chemotherapy sensitizer. Verapamil and resveratrol enhanced the effects of ursolic acid in both cell lines, whereas cyclosporin A only did so in Ca3/7 cells. Similarly, verapamil inhibited Rh123 efflux in both lines, whereas cyclosporin A only inhibited Rh123 efflux in Ca3/7 cells. Resveratrol did not inhibit Rh123 efflux in either line, indicating the synergistic effects of resveratrol and ursolic acid are not manifest by inhibition of P-gp–mediated efflux of ursolic acid. These results indicate that the anti-skin cancer effects of ursolic acid are enhanced with P-gp inhibitors. In addition, resveratrol and ursolic acid interact synergistically, but not through inhibition of P-gp. Implications: Resveratrol and/or p-glycoprotein inhibitors in combination with ursolic acid are an effective anti-skin cancer regimen. Mol Cancer Res; 11(12); 1521–9. ©2013 AACR.

Ursolic acid and resveratrol synergize with chloroquine to reduce melanoma cell viability

Malignant melanoma is associated with a 5-year survival rate of less than 20% once metastasized. Malignant melanoma cells exhibit increased levels of autophagy, a process of intracellular digestion that allows cells to survive various stresses including chemotherapies, resulting in reduced patient survival. Autophagy can be inhibited by chemicals like chloroquine (CQ), which prevents fusion of autophagosomes to lysosomes, resulting in autophagosome accumulation in most systems. Here, we describe how tested CQ to see whether it could sensitize B16F10 metastatic mouse melanoma cells to the anticancer activities of the natural compounds ursolic acid (UA) and resveratrol (RES). CQ with UA or RES strongly and synergistically reduced the viability of B16F10 mouse melanoma and A375 human melanoma cells. Surprisingly, flow cytometry of acridine orange-stained cells showed that UA or RES in combination with CQ significantly reduced autophagosome levels. Western blotting analysis revealed that CQ plus UA or RES paradoxically increased LC3II, indicative of autophagosome accumulation. In addition, CQ plus RES synergistically decreased the levels of both autophagy initiator beclin-1 and autophagy supporter p62. These results indicate that CQ with UA or RES strongly and synergistically reduces the viability of B16F10 and A375 melanoma cells. However, studies on B16F10 cells have shown that the synergistic effect was not mediated by inhibition of autophagy induced by UA or RES. These compounds are well-tolerated in humans, and CQ has shown promise as an adjuvant therapy. These combinations may be valuable treatment strategies for melanoma.

Differential effects on lung cancer cell proliferation by agonists of glucocorticoid and PPARα receptors

Glucocorticoids (GCs) are well‐known anti‐inflammatory compounds, but they also inhibit cell proliferation depending on cell type. Similarly, peroxisome proliferator‐activated receptors (PPARα, PPARδ, and PPARγ) also possess anti‐proliferation properties beyond their canonical roles as metabolic mediators. In the present study, we investigated the potential additive or synergistic inhibitory effects on cancer cell proliferation by simultaneous application of fenofibrate and budesonide, agonists for PPARα and glucocorticoid receptor, respectively. We observed differential effects on cell proliferation in A549 and SK‐MES‐1 lung cancer cells by budesonide and fenofibrate. Fenofibrate inhibited cell proliferation in both TP53 wild type and deficient lung cancer cells. The anti‐proliferation effect of budesonide in TP53 wild type A549 cells was abolished in SK‐MES‐1 cells that do not have wild type TP53 protein. An additive effect against cell proliferation by budesonide and fenofibrate combination was observed only in TP53 wild type A549 cancer cells. Analysis of cell cycle distribution and cyclin profile indicated that the inhibition of cell proliferation was associated with G1 cell cycle arrest. The suppression of NF‐κB activity and ERK signaling may contribute to the inhibition of cell proliferation by budesonide and or fenofibrate. The additive inhibitory effect on cell proliferation by budesonide and fenofibrate combination suggests that the same or greater therapeutic effect could be achieved with reduced dosage and side effects when the two compounds are applied simultaneously.

PGC-1α-induced mitochondrial alterations in 3T3 fibroblast cells

Abstract:  Peroxisome proliferation activator receptor (PPAR) γ‐coactivator 1α (PGC‐1α), a transcription coactivator, functions as a master regulator of a wide array of metabolic and physiological processes and is an essential factor in the process of mitochondrial biogenesis. Transfection of NIH 3T3 fibroblasts with a mouse cDNA for PGC‐1α led to the induction of markers of mitochondrial biogenesis, that is, mitochondrial transcription factor A (mtTFA), cytochrome c, and mitochondrial DNA (mtDNA). Mitochondrial biogenesis‐associated net protein synthesis appears to be accomplished by a reduction in the rate of mitochondrial protein degradation with little or no change in the rate of protein synthesis. Overexpression of PGC‐1α did not adversely affect cellular proliferation. Cellular ATP levels were increased in the transfected cells and they were more resistant to oxidative stress than the control nontransfected 3T3 cells. This resistance to oxidative stress was manifested by both an improved viability and the maintenance of mitochondrial membrane potential in the transfected cells when exposed to t‐butyl hydroperoxide (t‐BOOH). It therefore appears that PGC‐1α overexpression stimulates mitochondrial biogenesis in 3T3 cells making them more resistant to oxidative stressors.