Effect of the JAK2/STAT3 inhibitor SAR317461 on human glioblastoma tumorspheres

BackgroundThe STAT3 transcription factor is a major intracellular signaling protein and is frequently dysregulated in the most common and lethal brain malignancy in adults, glioblastoma multiforme (GBM). Activation of STAT3 in GBM correlates with malignancy and poor prognosis. The phosphorylating signal transducer JAK2 activates STAT3 in response to cytokines and growth factors. Currently there are no JAK-STAT pathway inhibitors in clinical trials for GBM, so we sought to examine the anti-GBM activity of SAR317461 (Sanofi-Aventis), a newer generation, highly potent JAK2 inhibitor that exhibits low toxicity and good pharmacokinetics. SAR317461 was initially approved for patient testing in the treatment of primary myelofibrosis (PMF), and has shown activity in preclinical models of melanoma and pulmonary cancer, but has not been tested in GBM.MethodsWe hypothesized that a potent small molecule JAK2 inhibitor could overcome the heterogeneous nature of GBM, and suppress a range of patient derived GBM tumorsphere lines and immortalized GBM cell lines. We treated with SAR317461 to determine IC50 values, and using Western blot analysis we asked whether the response was linked to STAT3 expression. Western blot analysis, FACS, and cell viability studies were used to identify the mechanism of SAR317461 induced cell death.ResultsWe report for the first time that the JAK2 inhibitor SAR317461 clearly inhibited STAT3 phosphorylation and had substantial activity against cells (IC50 1-10 µM) from 6 of 7 different patient GSC derived GBM tumorsphere lines and three immortalized GBM lines. One patient GSC derived line did not constitutively express STAT3 and was more resistant to SAR317461 (IC50 ≈25 µM). In terms of mechanism we found cleaved PARP and clear apoptosis following SAR317461. SAR317461 also induced autophagy and the addition of an autophagy inhibitor markedly enhanced cell killing by SAR317461.ConclusionsWe conclude that SAR317461 potently inhibits STAT3 phosphorylation and that it has significant activity against those GBM cells which express activated STAT3. Further studies are warranted in terms of the potential of SAR317461 as single and combined therapy for selectively treating human patients afflicted with GBMs expressing activation of the JAK2-STAT3 signaling axis

Allosteric inhibitor of β-catenin selectively targets oncogenic Wnt signaling in colon cancer.

Abnormal regulation of β-catenin initiates an oncogenic program that serves as a main driver of many cancers. Albeit challenging, β-catenin is an attractive drug target due to its role in maintenance of cancer stem cells and potential to eliminate cancer relapse. We have identified C2, a novel β-catenin inhibitor, which is a small molecule that binds to a novel allosteric site on the surface of β-catenin. C2 selectively inhibits β-catenin, lowers its cellular load and significantly reduces viability of β-catenin-driven cancer cells. Through direct binding to β-catenin, C2 renders the target inactive that eventually activates proteasome system for its removal. Here we report a novel pharmacologic approach for selective inhibition of β-catenin via targeting a cryptic allosteric modulation site. Our findings may provide a new perspective for therapeutic targeting of β-catenin

Downregulation of hepatic lipopolysaccharide binding protein improves lipogenesis-induced liver lipid accumulation

Circulating lipopolysaccharide-binding protein (LBP) is increased in individuals with liver steatosis. We aimed to evaluate the possible impact of liver LBP downregulation using lipid nanoparticle-containing chemically modified LBP small interfering RNA (siRNA) (LNP-Lbp UNA-siRNA) on the development of fatty liver. Weekly LNP-Lbp UNA-siRNA was administered to mice fed a standard chow diet, a high-fat and high-sucrose diet, and a methionine- and choline-deficient diet (MCD). In mice fed a high-fat and high-sucrose diet, which displayed induced liver lipogenesis, LBP downregulation led to reduced liver lipid accumulation, lipogenesis (mainly stearoyl-coenzyme A desaturase 1 [Scd1]) and lipid peroxidation-associated oxidative stress markers. LNP-Lbp UNA-siRNA also resulted in significantly decreased blood glucose levels during an insulin tolerance test. In mice fed a standard chow diet or an MCD, in which liver lipogenesis was not induced or was inhibited (especially Scd1 mRNA), liver LBP downregulation did not impact on liver steatosis. The link between hepatocyte LBP and lipogenesis was further confirmed in palmitate-treated Hepa1-6 cells, in primary human hepatocytes, and in subjects with morbid obesity. Altogether, these data indicate that siRNA against liver Lbp mRNA constitutes a potential target therapy for obesity-associated fatty liver through the modulation of hepatic Scd1

spacer-arm modulated gene delivery efficacy of novel cationic glycolipids: design, synthesis, and in vitro transfection biology

Design, syntheses and relative in vitro gene delivery efficacies of six novel cationic glycolipids 1-6 containing open-form galactosyl units in CHO, COS-1, MCF-7 and A549 cells are described. The results of the present structure-activity investigation convincingly demonstrate that the in vitro gene delivery efficacies of galactosylated cationic glycolipids are strikingly dependent on the absence of a spacer-arm between the open-form galactose and the positively charged nitrogen atom in their headgroup region. While the cationic glycolipids 1-3 with no headgroup spacer unit between the positively charged nitrogen and galactose showed high in vitro gene transfer efficacies in all four cells (lipids 1 and 2 with myristyl and palmityl tails, respectively, being the most efficacious), lipids 4-6 with five-carbon spacer units between the quaternized nitrogen and galactose heads were essentially transfection incompetent. The transfection inhibiting role of the five-carbon spacer unit in the headgroup region of the present novel class of cationic lipids was demonstrated by both β-galactosidase reporter gene expression and histochemical X-gal staining assays. Results of MTT assay-based cell viability measurements in representative MCF7 cells show that cell viabilities of lipoplexes (lipid:DNA complexes) prepared from all the lipids 1-6 are remarkably high. Thus, possibilities of differential cellular cytotoxicities playing any key role behind the strikingly contrasting transfection properties of lipids 1-3 with no spacer and lipids 4-6 with a spacer unit in the headgroup regions was ruled out. Electrophoresis gel patterns in DNase I sensitivity assays are consistent with more free DNA (accessible to DNase I) being present in lipoplexes of lipids 4-6 than in lipoplexes of lipids 1-3. Thus, the results of our DNase I protection experiments support the notion that enhanced degradation of DNA associated with lipoplexes of lipids 4-6 may play an important role in abolishing their in vitro gene transfer efficacies

Dramatic influence of the orientation of linker between hydrophilic and hydrophobic lipid moiety in liposomal gene delivery

A number of prior studies have demonstrated that the DNA-binding and gene transfection efficacies of cationic amphiphiles crucially depend on their various structural parameters including hydrophobic chain lengths, headgroup functionalities, and the nature of the linker-functionality used in tethering the polar headgroup and hydrophobic tails. However, to date addressing the issue of linker orientation remains unexplored in liposomal gene delivery. Toward probing the influence of linker orientation in cationic lipid mediated gene delivery, we have designed and synthesized two structurally isomeric remarkably similar cationic amphiphiles 1 and 2 bearing the same hydrophobic tails and the same polar headgroups connected by the same ester linker group. The only structural difference between the cationic amphiphiles 1 and 2 is the orientation of their linker ester functionality. While lipid 1 showed high gene transfer efficacies in multiple cultured animal cells, lipid 2 was essentially transfection incompetent. Findings in both transmission electron microscopic and dynamic laser light scattering studies revealed no significant size difference between the lipoplexes of lipids 1 and 2. Findings in confocal microscopic and fluorescence resonance energy transfer (FRET) experiments, taken together, support the notion that the remarkably higher gene transfer efficacies of lipid 1 compared to those of lipid 2 presumably originate from higher biomembrane fusogenicity of lipid 1 liposomes. Differential scanning calorimetry (DSC) and fluorescence anisotropy studies revealed a significantly higher gel-to-liquid crystalline temperature for the lipid 2 liposomes than that for lipid 1 liposomes. Findings in the dye entrapment experiment were also consistent with the higher rigidity of lipid 2/cholesterol (1:1 mole ratio) liposomes. Thus, the higher biomembrane fusibility of lipid 1 liposomes than that of lipid 2 liposomes presumably originates from the more rigid nature of lipid 2 cationic liposomes. Taken together, the present findings demonstrate for the first time that even as minor a structural variation as linker orientation reversal in cationic amphiphiles can profoundly influence DNA-binding characteristics, membrane rigidity, membrane fusibility, cellular uptake, and consequently gene delivery efficacies of cationic liposomes

Anti-cancer effects of nitrogen-containing bisphosphonates on human cancer cells.

Zoledronic acid, a potent nitrogen-containing bisphosphonate (NBP), has been extensively used to limit bone turnover in a various diseases including tumors. Recent clinical studies have demonstrated direct anti-cancer effects of zoledronic acid, in addition to its clinical benefits for skeletal-related events. Here we investigated the effects of 4 clinically available NBPs on human tumor cell proliferation. Our data demonstrate a potent anti-proliferative effect of zoledronic acid against glioblastoma (GBM) cell lines, breast cancer cells and GBM patient-derived lines. Zoledronic acid also effectively inhibited GBM tumor growth in xenograft mouse models. Zoledronic acid strongly stimulated autophagy but not apoptotic signals in all tested cells. Only one intermediate product of cholesterols synthesis pathway, geranylgeranyl diphosphate (GGPP) rescued cells from the cytotoxic effects of zoledronic acid. To further investigate the effect of GGPP, we knocked down RABGGTA, which encodes a subunit of the Rabgeranylgeranyltransferase protein. This knockdown induced an effect similar to zoledronic acid in cancer cell lines. These data are promising and suggested a potential for zoledronic acid as an anti-cancer agent, through its ablation of the function of Rab proteins