Giri, Shailendra

Abstract Multiple sclerosis is an inflammatory, demyelinating disease of the CNS of presumed autoimmune origin. Of all the genetic factors linked with multiple sclerosis, MHC class II molecules have the strongest association. Generation of HLA class II transgenic (Tg) mice has helped to elucidate the role of HLA class II genes in chronic inflammatory and demyelinating diseases. We have shown that the human HLA-DRB1*0301 gene predisposes to proteolipid protein (PLP)–induced experimental autoimmune encephalomyelitis (EAE), whereas HLA-DQβ1*0601 (DQ6) was resistant. We also showed that the DQ6 molecule protects from EAE in DRB1*0301.DQ6 double-Tg mice by producing anti-inflammatory IFN-γ. HLA-DQβ1*0302 (DQ8) Tg mice were also resistant to PLP91–110–induced EAE, but production of proinflammatory IL-17 exacerbated disease in DRB1*0301.DQ8 mice. To further confirm the role of IFN-γ in protection, we generated DRB1*0301.DQ8 mice lacking IFN-γ (DRB1*0301.DQ8.IFN-γ−/−). Immunization with PLP91–110 peptide caused atypical EAE in DRB1*0301.DQ8.IFN-γ−/− mice characterized by ataxia, spasticity, and dystonia, hallmarks of brain-specific disease. Severe brain-specific inflammation and demyelination in DRB1*0301.DQ8.IFN-γ−/− mice with minimal spinal cord pathology further confirmed brain-specific pathology. Atypical EAE in DRB1*0301.DQ8.IFN-γ−/− mice was associated with increased encephalitogenicity of CD4 T cells and their ability to produce greater levels of IL-17 and GM-CSF compared with DRB1*0301.DQ8 mice. Further, areas with demyelination showed increased presence of CD68+ inflammatory cells, suggesting an important role for monocytes/microglia in causing brain pathology. Thus, our study supports a protective role for IFN-γ in the demyelination of brain through downregulation of IL-17/GM-CSF and induction of neuroprotective factors in the brain by monocytes/microglial cells.

Significance Cancer cells reprogram their metabolism for optimal growth and survival. AMPK-activated protein kinase (AMPK) is a key energy sensor that controls many metabolic pathways including metabolic reprogramming. However, its role in cancer is poorly understood. Some studies claim that it has a tumor suppressor role while others show its protumor role. Two AMPK-activating compounds (including metformin, now in many clinical trials) are widely used to suppress cancer cell proliferation. We found that AMPK is abundantly expressed in high-grade gliomas and, in contrast to popular belief, these two AMPK activators suppressed glioma cell proliferation through unique AMPK-independent mechanisms.

Abstract PG545 is a synthetic heparan sulfate (HS) mimetic purported to inhibit angiogenesis via the blockade of VEGF and FGF-2 signaling, and block the enzymatic activity of heparanase - a molecule closely associated with metastatic dissemination. The attenuation of VEGF and FGF-2 signaling is supported by recent clinical data from the first four advanced cancer patients with multiple tumor types treated with PG545 which exhibited increases in plasma VEGF and FGF-2 levels, often in close association with the drug treatments/exposure profile during the first treatment cycle over 4 weeks. Unfortunately, due to local injection site reactions associated with subcutaneous (SC) administration, the trial was recently terminated. However, to support a re-entry to the clinic using the intravenous route, we tested PG545 in a preclinical model of ovarian cancer.The rationale to investigate PG545 in ovarian cancer is derived from recently generated data which found PG545 (5 µM) almost completely inhibited migration of SKOV3 ovarian carcinoma cells stimulated by each of the following HS binding growth factors: VEGF, HGF, FGF-2, SDF-1 and HB-EGF. Because HB-EGF is thought to play a major role in the spread of this cancer, PG545 was also tested against HB-EGF dependant SKOV3 cell invasion and was found to be a potent inhibitor of this process. The effect of PG545 on HB-EGF signaling pathways in these experiments were investigated and the results indicated that PG545 reduces activation of the HB-EGF receptor (EGFR) and, consequently, both AKTand MAPK signaling are also reduced. Given the strong mechanism of action data for ovarian carcinoma, PG545 was next tested in the A2780 ovarian xenograft model. The signficant antitumor activity using PG545 at 7.5 mg/kg twice weekly intravenous (IV) and 15 mg/kg once weekly IV was similar to a 20 mg/kg once weekly SC dose. Pharmacokinetic analysis confirmed that drug exposure (AUC) over the course of a week was consistent between groups. PG545 significantly reduced tumor burden in a cisplatin resistant ovarian tumor model (C200) when given intraperitoneally at the dose of 20mg/kg, once a week until the end of the study. Most importantly, PG545 completely reduced tumor mass and reduced ascites accumulation in a syngeneic immunocompetent mouse model of ovarian cancer (ID8). Given the known effects of PG545 on heparanase and in metastatic models, these emerging data on growth factors in the clinic and HB-EGF in preclinical models provide a strong rationale to re-enter the clinic using the IV route and to focus on ovarian cancer as the clinical indication. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-309. doi:1538-7445.AM2012-LB-309

Abstract A substantial body of evidence suggests an association between vascular endothelial growth factor (VEGF) and the neovascularization that occurs with ovarian carcinogenesis. VEGF expression has been directly related to ascites accumulation. Phase II clinical trials in ovarian cancer using single-agent bevacizumab (a monoclonal antibody directed against VEGF) showed promising results, clearly highlighting the importance of anti-angiogenesis based-targeted therapies in ovarian cancer. One of the ways that VEGF mediates angiogenesis is by activating NADPH oxidase-ROS pathway. We have developed and engineered nanoparticles (NP) of cerium oxide (Nanoceria, NCe), a rare earth oxides, which have been tested for their ability to serve as free-radical scavengers to provide protection against chemical, biological, and radiological insults that promote the production of free radicals. Here we show for the first time that treatment of human umbilical vascular endothelial cells (HUVECs) with NCe resulted in inhibition of proliferation, capillary tube formation and VEGF165 induced activation of VEGFR2 and MMP2. Additionally, NCe treatment inhibited growth factor induced migration and invasion of ovarian cancer cells. Collectively, these results indicate that cerium oxide based NP could potentially be used as an antiangiogenic therapeutic agent in ovarian cancer. Citation Information: Cancer Res 2009;69(23 Suppl):C48.

Abstract Metformin is the most widely used drug for type 2 diabetes. Metformin activates AMP-activated protein kinase (AMPK), which regulates cellular energy metabolism via its upstream kinase LKB1. Activation of AMPK has been shown extensively to cause growth arrest in multiple tumor types by inhibiting the TSC-mTOR (mammalian target of rapamycin) pathway as well as by inducing cell cycle arrest. Our data indicate that metformin exerts an anti-proliferative effect on several chemo-responsive and -resistant ovarian cancer cell lines (A2780, CP70, C200, OV202, OVCAR3 and SKVO3ip, PE01, PE04), which is accompanied by activation of AMPK and cell cycle arrest. Interestingly, we have also determined that metformin is able to inhibit proliferation of ovarian cancer cells when AMPK is silenced by siRNA, where both the expression and activation of AMPK is significantly reduced. Also, metformin was able to attenuate proliferation of AMPK (−/−) mefs, although to a lesser degree in AMPK (+/+) mefs. Additional results indicate that metformin activates p21 and inhibits cyclinD1 activities partially in AMPK (−/) mefs. These data suggest a novel mechanism of metformin action, namely, an AMPK independent pathway which can modulate proliferation and possibly cell cycle regulated events in ovarian cancer. Whether the AMPK independent effects of metformin require LKB1 activity is currently under investigation. Collectively, our preliminary observations indicate that metformin can modulate proliferation and cell cycle arrest in an AMPK independent manner. Citation Information: Cancer Res 2009;69(23 Suppl):C38.

Abstract Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the CNS. Metformin is the most widely used drug for diabetes and mediates its action via activating AMP-activated protein kinase (AMPK). We provide evidence that metformin attenuates the induction of EAE by restricting the infiltration of mononuclear cells into the CNS, down-regulating the expression of proinflammatory cytokines (IFN-γ, TNF-α, IL-6, IL-17, and inducible NO synthase (iNOS)), cell adhesion molecules, matrix metalloproteinase 9, and chemokine (RANTES). Furthermore, the AMPK activity and lipids alterations (total phospholipids and in free fatty acids) were restored by metformin treatment in the CNS of treated EAE animals, suggesting the possible involvement of AMPK. Metformin activated AMPK in macrophages and thereby inhibited biosynthesis of phospholipids as well as neutral lipids and also down-regulated the expression of endotoxin (LPS)-induced proinflammatory cytokines and their mediators (iNOS and cyclooxygenase 2). It also attenuated IFN-γ and IL-17-induced iNOS and cyclooxygenase 2 expression in RAW267.4 cells, further supporting its anti-inflammatory property. Metformin inhibited T cell-mediated immune responses including Ag-specific recall responses and production of Th1 or Th17 cytokines, while it induced the generation of IL-10 in spleen cells of treated EAE animals. Altogether these findings reveal that metformin may have a possible therapeutic value for the treatment of multiple sclerosis and other inflammatory diseases.

SummaryRecent reports show that 5-amino-4-imidazole carboxamide riboside (AICAR), a pharmacological activator of AMP-activated protein kinase (AMPK), inhibits the lipopolysaccharide (LPS)-induced production of proinflammatory cytokines. MRL/MPJ-Faslpr (MRL/lpr) mice show an intrinsic decreased threshold for the production of inflammatory mediators when stimulated. In our current studies, we sought to determine if AMPK activation would inhibit inflammatory mediator production in stimulated kidney mesangial cells. Cultured mesangial cells from MRL/lpr mice were treated with AICAR and stimulated with LPS/interferon (IFN)-γ. AICAR decreased dose-dependently inducible nitric oxide synthase (iNOS), cyclooxygenase-2 and interleukin-6 production in LPS/IFN-γ-stimulated mesangial cells. Mechanistically, AICAR inhibited the LPS/IFN-γ-stimulated PI3K/Akt signalling inflammatory cascade but did not affect LPS/IFN-γ-mediated inhibitory kappa B phosphorylation or nuclear factor (NF)-κB (p65) nuclear translocation. Treatment with the adenosine kinase inhibitor 5′-iodotubercidin blocked the ability of AICAR to activate AMPK and prevented AICAR from inhibiting the LPS/IFN-γ-stimulated PI3K/Akt pathway and attenuating iNOS expression. Taken together, these observations suggest that AICAR inhibits LPS/IFN-γ-induced Akt phosphorylation through AMPK activation and may serve as a potential therapeutic target in inflammatory diseases.