Dual Beneficial Effects of Methylnissolin-3-O-ß-d-Glucopyranoside on Obesity-Induced Inflammatory Responses in Adipocyte-Macrophage Co-Culture.

Methylnissolin-3-O-ß-d-glucopyranoside (MNG) is a pterocarpan analog, which protects EA.hy926 cells against oxidative damage through the Nrf2/HO-1 pathway. However, the effects of MNG on obesity-induced inflammatory responses in adipocyte-macrophage co-culture remain unclear. A differentiated murine preadipocyte cell line (3T3-L1) was co-cultured with a murine macrophage cell line (RAW264.7). Intracellular lipid accumulation was determined using Oil Red O staining. Western blotting was performed to investigate the expression of adipogenesis- and inflammation-associated proteins. Cell culture supernatants were assayed using ELISA kits to measure the levels of proinflammatory cytokines such as interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). MNG inhibited lipid accumulation and the production of IL-6 and MCP-1 in the 3T3-L1 and RAW264.7 cell co-culture. Moreover, MNG inhibited the protein expression of CCAAT/enhancer-binding protein alpha (C/EBPα), C/EBPß, peroxisome proliferator-activated receptor γ (PPARγ), cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS) under the same co-culture conditions. MNG also inhibited IL-6 and MCP-1 production compared with the co-culture control. These findings demonstrate that MNG inhibited lipid accumulation and inflammatory response by downregulating IL-6 and MCP-1 production and protein expression of C/EBPß, C/EBPα, PPARγ, COX-2, and iNOS in co-culture conditions with 3T3-L1 and RAW264.7 cells. These results suggest that MNG may be beneficial in preventing obesity-related inflammatory status.

Synthesis and Characterization of Lipophilic Salts of Metformin to Improve Its Repurposing for Cancer Therapy.

Epidemiological evidence has accentuated the repurposing of metformin hydrochloride for cancer treatment. However, the extreme hydrophilicity and poor permeability of metformin hydrochloride are responsible for its poor anticancer activity in vitro and in vivo. Here, we report the synthesis and characterization of several lipophilic metformin salts containing bulky anionic permeation enhancers such as caprate, laurate, oleate, cholate, and docusate as counterions. Of various counterions tested, only docusate was able to significantly improve the lipophilicity and lipid solubility of metformin. To evaluate the impact of the association of anionic permeation enhancers with metformin, we checked the in vitro anticancer activity of various lipophilic salts of metformin using drug-sensitive (MYCN-2) and drug-resistant (SK-N-Be2c) neuroblastoma cells as model cancer cells. Metformin hydrochloride showed a very low potency (IC50 ≈ >100 mM) against MYCN-2 and SK-N-Be2c cells. Anionic permeation enhancers showed a considerably higher activity (IC50 ≈ 125 µM to 1.6 mM) against MYCN-2 and SK-N-Be2c cells than metformin. The association of metformin with most of the bulky anionic agents negatively impacted the anticancer activity against MYCN-2 and SK-N-Be2c cells. However, metformin docusate showed 700- to 4300-fold improvement in anticancer potency compared to metformin hydrochloride and four- to five-fold higher in vitro anticancer activity compared to sodium docusate, indicating a synergistic association between metformin and docusate. A similar trend was observed when we tested the in vitro activity of metformin docusate, sodium docusate, and metformin hydrochloride against hepatocellular carcinoma (HepG2) and triple-negative breast cancer (MDA-MB-231) cells.

In vitro efficacy of anthelmintics on Angiostrongylus cantonensis L3 larvae.

Angiostrongylus cantonensis is the leading cause of eosinophilic meningitis worldwide, with life-threatening complications if not managed correctly. Previous in vitro studies have utilized change in motility patterns of adult female worms to assess the efficacy of anthelmintics qualitatively. However, it is the third stage larvae (L3) that are infectious to humans. With differential staining using propidium iodide penetration as the indicator of death, we can distinguish between dead and live larvae. This assay has enabled us to quantify the in vitro efficacy of nine clinically established anthelmintics on A. cantonensis L3. All drugs were tested at a 1 mm concentration. Piperazine and niclosamide were ineffective in inducing larval death; however, albendazole sulfoxide, pyrantel pamoate, diethylcarbamazine, levamisole and praziquantel were effective as compared to unexposed controls (P < 0.05). Ivermectin and moxidectin did not induce significant levels of mortality, but they considerably reduced larval motility almost immediately. This study indicates the need for further in vivo studies to determine the optimal dose and time frame for post-infection treatment with anthelmintics that demonstrated efficacy.

Validation of a death assay for Angiostrongylus cantonensis larvae (L3) using propidium iodide in a rat model (Rattus norvegicus).

Angiostrongylus cantonensis is a pathogenic nematode and the cause of neuroangiostrongyliasis, an eosinophilic meningitis more commonly known as rat lungworm disease. Transmission is thought to be primarily due to ingestion of infective third stage larvae (L3) in gastropods, on produce, or in contaminated water. The gold standard to determine the effects of physical and chemical treatments on the infectivity of A. cantonensis L3 larvae is to infect rodents with treated L3 larvae and monitor for infection, but animal studies are laborious and expensive and also raise ethical concerns. This study demonstrates propidium iodide (PI) to be a reliable marker of parasite death and loss of infective potential without adversely affecting the development and future reproduction of live A. cantonensis larvae. PI staining allows evaluation of the efficacy of test substances in vitro, an improvement upon the use of lack of motility as an indicator of death. Some potential applications of this assay include determining the effectiveness of various anthelmintics, vegetable washes, electromagnetic radiation and other treatments intended to kill larvae in the prevention and treatment of neuroangiostrongyliasis.

Calcium signaling regulates fundamental processes involved in Neuroblastoma progression.

Neuroblastoma (NB) is the most common extra-cranial pediatric solid tumor in children. Despite NB's relative rarity, high-risk NB patients have a poor prognosis with survival rate less than 50%. This is even worse for patients with relapsed or refractory NB. Finding effective alternative treatment strategies is therefore a must. Calcium is an intracellular messenger that is unequivocally present in normal physiology mediating proliferation, growth, migration, cell division, angiogenesis and cell death, as well as pathophysiological processes such as those included in Weinberg's hallmarks of cancer. Within the past 20 years, the molecular identity of most calcium channels has been revealed, however for some of these channels the precise gating mechanism and their role in normal physiology is still elusive. Here we review the recent findings of components of calcium signaling that are deregulating in the malignant progression of NB. We further integrate critical calcium signaling pathways using patient-derived expression analysis. Revealing the roles of these calcium pathways in tumor development, progression, microenvironment and importantly - protection against antineoplastic drugs may hopefully lead to novel treatment strategies in the future.

Potential anti-neuroblastoma agents from Juniperus oblonga.

Neuroblastoma (NB) is a neuroendocrine tumor derived from neural crest cells. Approximately 90% of cases occur in children less than 5 years old. The amplification of MYCN correlates with high-risk neuroblastoma and patients with MYCN amplified showed poorer prognosis than those without MYCN amplification. In this study, three compounds isolated from Juniperus oblonga showed anti-proliferative activity against NB cell lines with and without tetracycline inducible MYCN over-expression which were identified as (-)-deoxypodophyllotoxin (1), (-)-matairesinol (2) and (+)-isocupressic acid (3). The effects of compounds 2 and 3 in NB cells included a decrease in NB cell viability and induction of apoptosis. Compound 1 was more effective in NB cells over-expressing MycN. Compound 1 also showed almost 2-fold induction of intracellular free calcium levels in M2(+) cells, which may indicate a different mechanism of action for this compound. Cytotoxicity studies against the human embryonic kidney cell (HEK-293) showed compounds 1, 2 and 3 were ineffective in the non-cancer cells at concentrations approximating their IC50 against the NB cell lines. These results may lead to safer and more effective treatment options for NB patients especially for those with high-risk NB.

STIM1 and STIM2 Mediate Cancer-Induced Inflammation in T Cell Acute Lymphoblastic Leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is commonly associated with activating mutations in the NOTCH1 pathway. Recent reports have shown a link between NOTCH1 signaling and intracellular Ca2+ homeostasis in T-ALL. Here, we investigate the role of store-operated Ca2+ entry (SOCE) mediated by the Ca2+ channel ORAI1 and its activators STIM1 and STIM2 in T-ALL. Deletion of STIM1 and STIM2 in leukemic cells abolishes SOCE and significantly prolongs the survival of mice in a NOTCH1-dependent model of T-ALL. The survival advantage is unrelated to the leukemic cell burden but is associated with the SOCE-dependent ability of malignant T lymphoblasts to cause inflammation in leukemia-infiltrated organs. Mice with STIM1/STIM2-deficient T-ALL show a markedly reduced necroinflammatory response in leukemia-infiltrated organs and downregulation of signaling pathways previously linked to cancer-induced inflammation. Our study shows that leukemic T lymphoblasts cause inflammation of leukemia-infiltrated organs that is dependent on SOCE.

FTY-720 induces apoptosis in neuroblastoma via multiple signaling pathways.

Neuroblastoma (NB) is the most common extra-cranial pediatric solid tumor. High-risk NB is difficult to treat due to the lack of response to current therapies and aggressive disease progression. Despite novel drugs, alternative treatments and multi-modal treatments, finding an effective treatment strategy for these patients continues to be a major challenge. The current study focuses on examining the effects of FTY-720 or fingolimod, a drug that is FDA-approved for refractory multiple sclerosis, in NB. The results showed that FTY-720 regulates multiple pathways that result in various effects on calcium signaling, ion channel activation and cell survival/death pathways. FTY-720 rapidly inhibits TRPM7 channel activity, and inhibited TRPM7 kinase activity, modulates calcium signaling, induces a loss of mitochondrial membrane potential and opening of the mitochondrial permeability transition pore, and ultimately leads to cell death. Interestingly, the data also showed that low concentrations of FTY-720 sensitized drug-resistant NB cells to antineoplastic drugs. These results suggest that FTY-720 may be an attractive alternative for the treatment of NB.

Scrophularia orientalis extract induces calcium signaling and apoptosis in neuroblastoma cells.

Effective neuroblastoma (NB) treatments are still limited despite treatment options available today. Therefore, this study attempted to identify novel plant extracts that have anticancer effects. Cytotoxicity and increased intracellular calcium levels were determined using the Sulforhodamine B (SRB) assay and Fluo4-AM (acetoxymethyl) staining and fluorescence microscopy in NB cells in order to screen a library of plant extracts. The current study examined the anticancer effects of a dichloromethane extract from Scrophularia orientalis L. (Scrophulariaceae), a plant that has been used in Traditional Chinese Medicine. This extract contained highly potent agents that significantly reduced cell survival and increased calcium levels in NB cells. Further analysis revealed that cell death induced by this extract was associated with intracellular calcium release, opening of the MPTP, caspase 3- and PARP-cleavage suggesting that this extract induced aberrant calcium signaling that resulted in apoptosis via the mitochondrial pathway. Therefore, agents from Scrophularia orientalis may have the potential to lead to new chemo-therapeutic anticancer drugs. Furthermore, targeting intracellular calcium signaling may be a novel strategy to develop more effective treatments for NB.

MycN promotes TRPM7 expression and cell migration in neuroblastoma through a process that involves polyamines.

Neuroblastoma is an extra-cranial solid cancer in children. MYCN gene amplification is a prognostic indicator of poor outcome in neuroblastoma. Recent studies have shown that the multiple steps involved in cell migration are dependent on the availability of intracellular calcium (Ca(2+)). Although significant advances have been made in understanding the role of Ca(2+) during migration, little has been achieved towards understanding its impact on the progression of diseases such as cancer. Interestingly, previous studies showed that cancer cell migration is regulated by TRPM7, a calcium-permeable ion channel. The objective of the current study was to elucidate the mechanism by which MycN promotes NB cell migration and the mechanism regulating TRPM7 expression. The results showed that MycN increased TRPM7 expression, induced TRPM7 channel activity, increased intracellular Ca(2+) signaling, and promoted cell migration in NB cells. The results also showed that inhibition or down-regulation of ornithine decarboxylase (ODC) inhibited TRPM7 expression, a process that was reversed by spermidine. Overall, this study provides evidence that MycN promotes TRPM7 expression and cell migration through a mechanism that involves ODC synthesis of polyamines.

Novel interaction of ornithine decarboxylase with sepiapterin reductase regulates neuroblastoma cell proliferation.

Ornithine decarboxylase (ODC) is the sentinel enzyme in polyamine biosynthesis. Both ODC and polyamines regulate cell division, proliferation, and apoptosis. Sepiapterin reductase (SPR) catalyzes the last step in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor of nitric oxide synthase, and has been implicated in neurological diseases but not yet in cancer. In this study, we present compelling evidence that native ODC and SPR physically interact, and we defined the individual amino acid residues involved in both enzymes using in silico protein-protein docking simulations. The resulting heterocomplex is a surprisingly compact structure, featuring two energetically and structurally equivalent binding modes both in monomer and in dimer conformations. The novel interaction between ODC and SPR proteins was confirmed under physiological conditions by co-immunoprecipitation and co-localization in neuroblastoma (NB) cells. Importantly, we showed that siRNA (small interfering RNA)-mediated knockdown of SPR expression significantly reduced endogenous ODC enzyme activity in NB cells, thus demonstrating the biological relevance of the ODC-SPR interaction. Finally, in a cohort of 88 human NB tumors, we found that high SPR mRNA expression correlated significantly with poor survival prognosis using a Kaplan-Meier analysis (log-rank test, P=5 × 10(-4)), suggesting an oncogenic role for SPR in NB tumorigenesis. In conclusion, we showed that ODC binds SPR and thus propose a new concept in which two well-characterized biochemical pathways converge via the interaction of two enzymes. We identified SPR as a novel regulator of ODC enzyme activity and, based on clinical evidence, present a model in which SPR drives ODC-mediated malignant progression in NB.

DFMO/eflornithine inhibits migration and invasion downstream of MYCN and involves p27Kip1 activity in neuroblastoma.

Neuroblastoma (NB) is the most common extracranial pediatric tumor. NB patients over 18 months of age at the time of diagnosis are often in the later stages of the disease, present with widespread dissemination, and often possess MYCN tumor gene amplification. MYCN is a transcription factor that regulates the expression of a number of genes including ornithine decarboxylase (ODC), a rate-limiting enzyme in the biosynthesis of polyamines. Inhibiting ODC in NB cells produces many deleterious effects including G(1) cell cycle arrest, inhibition of cell proliferation, and decreased tumor growth, making ODC a promising target for drug interference. DFMO treatment leads to the accumulation of the cyclin-dependent kinase inhibitor p27(Kip1) protein and causes p27(Kip1)/Rb-coupled G(1) cell cycle arrest in MYCN-amplified NB tumor cells through a process that involves p27(Kip1) phosphorylation at residues Ser10 and Thr198. While p27(Kip1) is well known for its role as a cyclin-dependent kinase inhibitor, recent studies have revealed a novel function of p27(Kip1) as a regulator of cell migration and invasion. In the present study we found that p27(Kip1) regulates the migration and invasion in NB and that these events are dependent on the state of phosphorylation of p27(Kip1). DFMO treatments induced MYCN protein downregulation and phosphorylation of Akt/PKB (Ser473) and GSK3-ß (Ser9), and polyamine supplementation alleviated the DFMO-induced effects. Importantly, we provide strong evidence that p27(Kip1) mRNA correlates with clinical features and the survival probability of NB patients.

Dendritic cell maturation and chemotaxis is regulated by TRPM2-mediated lysosomal Ca2+ release.

Chemokines induce calcium (Ca(2+)) signaling and chemotaxis in dendritic cells (DCs), but the molecular players involved in shaping intracellular Ca(2+) changes remain to be characterized. Using siRNA and knockout mice, we show that in addition to inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release and store-operated Ca(2+) entry (SOCE), the transient receptor potential melastatin 2 (TRPM2) channel contributes to Ca(2+) release but not Ca(2+) influx in mouse DCs. Consistent with these findings, TRPM2 expression in DCs is restricted to endolysosomal vesicles, whereas in neutrophils, the channel localizes to the plasma membrane. TRPM2-deficient DCs show impaired maturation and severely compromised chemokine-activated directional migration as well as bacterial-induced DC trafficking to the draining lymph nodes. Defective DC chemotaxis is due to perturbed chemokine-receptor-initiated Ca(2+) signaling mechanisms, which include suppression of TRPM2-mediated Ca(2+) release and secondary modification of SOCE. DCs deficient in both TRPM2 and IP(3) receptor signaling lose their ability to perform chemotaxis entirely. These results highlight TRPM2 as a key player regulating DC chemotaxis through its function as Ca(2+) release channel and confirm ADP-ribose as a novel second messenger for intracellular Ca(2+) mobilization.

Protein kinase C-induced phosphorylation of Orai1 regulates the intracellular Ca2+ level via the store-operated Ca2+ channel.

Ca(2+) signals through store-operated Ca(2+) (SOC) channels, activated by the depletion of Ca(2+) from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca(2+) sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca(2+) entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKCbeta both resulted in increased Ca(2+) influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30.

TRPM2 functions as a lysosomal Ca2+-release channel in beta cells.

TRPM2 is a Ca2+-permeable cation channel that is specifically activated by adenosine diphosphoribose (ADPR). Channel activation in the plasma membrane leads to Ca2+ influx and has been linked to apoptotic mechanisms. The primary agonist, ADPR, is produced both extra- and intracellularly and causes increases in intracellular calcium concentration ([Ca2+]i), but the mechanisms involved are not understood. Using short interfering RNA and a knockout mouse, we report that TRPM2, in addition to its role as a plasma membrane channel, also functions as a Ca2+-release channel activated by intracellular ADPR in a lysosomal compartment. We show that both functions of TRPM2 are critically linked to hydrogen peroxide-induced beta cell death. Additionally, extracellular ADPR production by the ectoenzyme CD38 from its substrates NAD+ (nicotinamide adenine dinucleotide) or cADPR causes IP3-dependent Ca2+ release via P2Y and adenosine receptors. Thus, ADPR and TRPM2 represent multimodal signaling elements regulating Ca2+ mobilization in beta cells through membrane depolarization, Ca2+ influx, and release of Ca2+ from intracellular stores.

A minimal regulatory domain in the C terminus of STIM1 binds to and activates ORAI1 CRAC channels.

Store-operated Ca(2+) entry (SOCE) is a universal mechanism to increase intracellular Ca(2+) concentrations in non-excitable cells. It is initiated by the depletion of ER Ca(2+) stores, activation of stromal interaction molecule (STIM) 1 and gating of the Ca(2+) release activated Ca(2+) (CRAC) channel ORAI1 in the plasma membrane. We identified a minimal activation domain in the cytoplasmic region of STIM1 (CCb9) which activated Ca(2+) influx and CRAC currents (I(CRAC)) in the absence of store depletion similar to but more potently than the entire C terminus of STIM1. A STIM1 fragment (CCb7) that is longer by 39 [corrected] amino acids than CCb9 at its C terminal end showed reduced ability to constitutively activate I(CRAC) consistent with our observation that CCb9 but not CCb7 efficiently colocalized with and bound to ORAI1. Intracellular application of a 31 amino acid peptide contained in CCb7 but not CCb9 inhibited constitutive and store-dependent CRAC channel activation. In summary, these findings suggest that CCb9 represents a minimal ORAI1 activation domain within STIM1 that is masked by an adjacent 31 amino acid peptide preventing efficient CRAC channel activation in cells with replete Ca(2+) stores.

TRPM2-mediated Ca2+influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration.

Reactive oxygen species (ROS) induce chemokines responsible for the recruitment of inflammatory cells to sites of injury or infection. Here we show that the plasma membrane Ca(2+)-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes. In human U937 monocytes, hydrogen peroxide (H(2)O(2)) evokes Ca(2+) influx through TRPM2 to activate Ca(2+)-dependent tyrosine kinase Pyk2 and amplify Erk signaling via Ras GTPase. This elicits nuclear translocation of nuclear factor-kappaB essential for the production of the chemokine interleukin-8 (CXCL8). In monocytes from Trpm2-deficient mice, H(2)O(2)-induced Ca(2+) influx and production of the macrophage inflammatory protein-2 (CXCL2), the mouse CXCL8 functional homolog, were impaired. In the dextran sulfate sodium-induced colitis inflammation model, CXCL2 expression, neutrophil infiltration and ulceration were attenuated by Trpm2 disruption. Thus, TRPM2 Ca(2+) influx controls the ROS-induced signaling cascade responsible for chemokine production, which aggravates inflammation. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases.

Synergistic regulation of endogenous TRPM2 channels by adenine dinucleotides in primary human neutrophils.

The Ca(2+)-permeable TRPM2 channel is a dual function protein that is activated by intracellular ADPR through its enzymatic pyrophosphatase domain with Ca(2+) acting as a co-factor. Other TRPM2 regulators include cADPR, NAADP and H(2)O(2), which synergize with ADPR to potentiate TRPM2 activation. Although TRPM2 has been thoroughly characterized in overexpression or cell-line systems, little is known about the features of TRPM2 in primary cells. We here characterize the regulation of TRPM2 activation in human neutrophils and report that ADPR activates TRPM2 with an effective half-maximal concentration (EC(50)) of 1microM. Potentiation by Ca(2+) is dose-dependent with an EC(50) of 300nM. Both cADPR and NAADP activate TRPM2, albeit with lower efficacy than in the presence of subthreshold levels of ADPR (100nM), which significantly shifts the EC(50) for cADPR from 44 to 3muM and for NAADP from 95 to 1microM. TRPM2 activation by ADPR can be suppressed by AMP with an IC(50) of 10microM and cADPR-induced activation can be blocked by 8-Bromo-cADPR. We further show that 100microM H(2)O(2) enables subthreshold concentrations of ADPR (100nM) to activate TRPM2. We conclude that agonistic and antagonistic characteristics of TRPM2 as seen in overexpression systems are largely compatible with the functional properties of TRPM2 currents measured in human neutrophils, but the potencies of agonists in primary cells are significantly higher.