Phenylboronic acid is a more potent inhibitor than boric acid of key signaling networks involved in cancer cell migration

Previous studies from our lab have shown that both boric (BA) and phenylboronic- acid (PBA) inhibit the migration of prostate cancer cell lines, as well as non-tumorigenic prostate cells. Our results indicate that PBA is more potent than BA in targeting metastatic and proliferative properties of cancer cells. Here we focus on the impact of BA and PBA on Rho family of GTP-binding proteins and their downstream targets. Treatment with 1mM PBA and BA decreases activities of RhoA, Rac1, and Cdc42 in DU-145 metastatic prostate cancer cells, but not in normal RWPE-1 prostate cells. Furthermore, ROCKII activity and phosphorylation of myosin light chain kinase decrease as a result of either PBA or BA treatment in DU-145 cells, suggesting these compounds target actomyosin-based contractility.     

R-etodolac is a more potent Wnt signaling inhibitor than enantiomer,S-etodolac

Etodolac is an FDA-approved nonsteroidal anti-inflammatory drug (NSAID) used to treat a variety of inflammatory diseases. The drug is administered as a racemate (50/50 mixture of R- and S- enantiomers), however, studies have shown that the two enantiomers have distinct biologic and pharmacokinetic differences. Wnt signaling, which plays key roles in cell proliferation, polarity, and differentiation, has been shown to be inhibited by R-etodolac; however, comparative analyses of R- and S-etodolac in this function have not been conducted. We used in silico molecular docking and TOPflash functional biologic assays to compare R- and S-enantiomers effect on Wnt signaling inhibition. Further, we used a cultivated limbal stem epithelial cell (cLSCs) model to investigate enantiospecific changes in the colony-forming efficiency (CFE) of cLSCs. The data shows that R-etodolac is a more potent inhibitor of Wnt signaling. In addition, consistently, while both enantiomers demonstrate a dose-dependent decrease in CFE of cLSCs, R-etodolac is a more potent inhibitor.     

Phenylboronic acid selectively inhibits human prostate and breast cancer cell migration and decreases viability

We compared the in vitro effect of boric acid (BA) versus phenylboronic acid (PBA) on the migration of prostate and breast cancer cell lines and non-tumorigenic cells from the same tissues. Treatment at 24 hours with BA (≤ 500 µM) did not inhibit chemotaxis on fibronectin in any cell line. However, treatment over the same time course with concentrations of PBA as low as 1 µM significantly inhibited cancer cell migration without effecting non-tumorigenic cell lines. The compounds did not affect cell adhesion or viability at 24 hours but did alter morphology; both decreased cancer cell viability at 8 days. These results suggest that PBA is more potent than BA in targeting the metastatic and proliferative properties of cancer cells.     

Chebulagic acid is a potent alpha-glucosidase inhibitor

Chebulagic acid, isolated form Terminalia chebula Retz, proved to be a reversible and non-competitive inhibitor of maltase with a K(i) value of 6.6 muM. The inhibitory influence of chebulagic acid on the maltase-glucoamylase complex was more potent than on the sucrase-isomaltase complex. The magnitude of alpha-glucosidase inhibition by chebulagic acid was greatly affected by its origin. These results show a use for chebulagic acid in managing type-2 diabetes.     

Conjugated nonadecadienoic acid is more potent than conjugated linoleic acid on body fat reduction

Conjugated linoleic acid (CLA) has shown a number of health benefits, particularly on controlling body fat while improving lean mass. As one of CLA cognates, conjugated nonadecadienoic acid (CNA, 19-carbon conjugated fatty acid) has been previously reported to have greater efficacy on body fat control. In this report, we compared the efficacy of dietary CLA and CNA on body fat regulation and also compared the mechanism of body fat control using a mouse model. Effects of 0.1% dietary CNA on body fat reduction were comparable to that of 0.5% dietary CLA. The mechanisms of dietary CNA on body fat control were similar to those of CLA: increased energy expenditure and increased fatty acid β-oxidation. Dietary CNA, but not CLA, also improved expression of hormone-sensitive lipase from white adipose tissue, and this may help explain how CNA has better efficacy on body fat control than CLA. Dietary CNA had similar effects as CLA on liver weights; however, unlike CLA, CNA improved glucose tolerance. Thus, CNA has potential to be used as a pharmacological agent to assist current efforts to reduce obesity with less adverse effects than CLA.     

Aurintricarboxylic acid is a potent inhibitor of phosphofructokinase.

Aurintricarboxylic acid (ATA) was found to be a very potent inhibitor of purified rabbit liver phosphofructokinase (PFK), giving 50% inhibition at 0.2 microM. The inhibition was in a manner consistent with interaction at the citrate-inhibitory site of the enzyme. The data suggest that inhibition of PFK by ATA was not due to denaturation of the enzyme or the irreversible binding of inhibitor, since the inhibition could be reversed by addition of allosteric activators of PFK, i.e. fructose 2,6-bisphosphate or AMP. Two other tricarboxylic acids, agaric acid and (-)-hydroxycitrate, were found to inhibit PFK. ATA at much higher concentrations (500 microM) was shown to inhibit fatty acid synthesis from endogenous glycogen in rat hepatocytes; however, protein synthesis was not altered.     

Ascorbic acid is a potent inhibitor of various forms of T cell apoptosis.

This study was designed to examine the effect of ascorbic acid (vitamin C) on various death pathways of mouse T cells. Unlike humans, mice produce their own ascorbic acid and our study tested the effect of additional ascorbic acid on murine T cells. Our data show that three T cell death pathways (growth factor withdrawal-, spontaneous-, and steroid-induced death) were inhibited when T cells were incubated with ascorbic acid. The data show that both activated and resting T cells were responsive to ascorbic acid since both populations were resistant to death stimuli when treated with ascorbic acid. Additionally, effector T cells were more likely to enter S phase if treated with ascorbic acid. Our data implicate ascorbic acid as a potent inhibitor of various forms of T cell death and suggest that vitamin C may function as an immune booster through this mechanism.     

Yucasin is a potent inhibitor of YUCCA,a key enzyme in auxin biosynthesis

Indole‐3–acetic acid (IAA), an auxin plant hormone, is biosynthesized from tryptophan. The indole‐3–pyruvic acid (IPyA) pathway, involving the tryptophan aminotransferase TAA1 and YUCCA (YUC) enzymes, was recently found to be a major IAA biosynthetic pathway in Arabidopsis. TAA1 catalyzes the conversion of tryptophan to IPyA, and YUC produces IAA from IPyA. Using a chemical biology approach with maize coleoptiles, we identified 5–(4–chlorophenyl)‐4H‐1,2,4–triazole‐3–thiol (yucasin) as a potent inhibitor of IAA biosynthesis in YUC‐expressing coleoptile tips. Enzymatic analysis of recombinant AtYUC1‐His suggested that yucasin strongly inhibited YUC1‐His activity against the substrate IPyA in a competitive manner. Phenotypic analysis of Arabidopsis YUC1 over‐expression lines (35S::YUC1) demonstrated that yucasin acts in IAA biosynthesis catalyzed by YUC. In addition, 35S::YUC1 seedlings showed resistance to yucasin in terms of root growth. A loss‐of‐function mutant of TAA1, sav3–2, was hypersensitive to yucasin in terms of root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor l –kynurenine acted additively in Arabidopsis seedlings, producing a phenotype similar to yucasin‐treated sav3–2 seedlings, indicating the importance of IAA biosynthesis via the IPyA pathway in root growth and leaf vascular development. The present study showed that yucasin is a potent inhibitor of YUC enzymes that offers an effective tool for analyzing the contribution of IAA biosynthesis via the IPyA pathway to plant development and physiological processes.     

The rapamycin analog CCI-779 is a potent inhibitor of pancreatic cancer cell proliferation

We present immunohistochemical evidence that the mTOR/p70s6k pathway is activated in pancreatic tumors and show that the mTOR inhibitor and rapamycin analog CCI-779 potently suppresses the proliferation of pancreatic cancer cells. Consistent with a recent study, CCI-779 increased c-Jun phosphorylation (Ser63) in a dose- and time-dependent manner, and induced apoptosis in p53-defective BxPC-3 cells. In contrast to the study, however, we observed that CCI-779 concomitantly increased c-Jun protein levels and that its ability to induce apoptosis might not require the activated c-Jun. Furthermore, CCI-779 neither induced c-Jun phosphorylation in other p53-defective pancreatic cancer cells (MiaPaCa-2) nor inhibited their proliferation. c-Jun, in fact, appeared to be partly responsible for the resistance of MiaPaCa-2 cells to CCI-779. Together, these results indicate a complex role for c-Jun in cellular responses to CCI-779 and provide an important basis for investigating CCI-779 further as a potential therapeutic agent for pancreatic tumors.     

Enamel matrix derivative is a potent inhibitor of breast cancer cell attachment to bone

This study examined whether enamel matrix derivative (EMD) inhibits the adhesion of cancer cells to bone. A typical breast cancer cell line, MCF-7, was used. Conditioned human osteosarcoma cell (Saos-2) medium was used as extracellular bone matrix (ECBM) to measure cell attachment. MCF-7 cells were incubated on ECBM-coated culture plates with or without soluble EMD, Arg-Gly-Asp (RGD) sequence blocking peptides, recombinant bone sialoprotein (rBSP), or specific integrin antibodies, and the attached cells were quantified using toluidine blue staining. EMD markedly reduced the attachment of MCF-7 cells to ECBM in a dose-dependent manner. An RGD peptide (GRGDSP) and recombinant BSP inhibited cell attachment to the same degree as EMD. Similarly, anti-alphavbeta3 integrin antibody strongly reduced cell attachment, whereas anti-alphavbeta5 and anti-beta1 integrin antibodies had less marked effects on cell attachment. These results show that EMD inhibits MCF-7 cell attachment to a bone matrix and that it might be useful as an anti-adhesive agent for breast cancer cells to bone in vivo.     

Homocysteine is a potent inhibitor of human tumor cell gelatinases

Extracellular matrix-degrading gelatinases are mainly involved in tumor invasion and metastasis. Previous experimental data from our group and others suggested that homocysteine could have a potential modulatory role on the proteolytic balance at the extracellular matrix. Therefore, we studied the effects of homocysteine on extracellular matrix-degrading proteases using model human tumor cell lines and a combination of in vitro fluorogenic assay and zymographic techniques. Homocysteine is shown to be the thiol compound with the most potent inhibitory activity on matrix metalloproteinase 9. Zymographies reveal that matrix metalloproteinase 2 is, at least, as sensitive to inhibition by homocysteine as matrix metalloproteinase 9 is. This study opens new ways to the potential pharmacological use of thiol compounds.     

Calcium is a key signaling molecule in beta-lapachone-mediated cell death

beta-Lapachone (beta-Lap) triggers apoptosis in a number of human breast and prostate cancer cell lines through a unique apoptotic pathway that is dependent upon NQO1, a two-electron reductase. Downstream signaling pathway(s) that initiate apoptosis following treatment with beta-Lap have not been elucidated. Since calpain activation was suspected in beta-Lap-mediated apoptosis, we examined alterations in Ca(2+) homeostasis using NQO1-expressing MCF-7 cells. beta-Lap-exposed MCF-7 cells exhibited an early increase in intracellular cytosolic Ca(2+), from endoplasmic reticulum Ca(2+) stores, comparable to thapsigargin exposures. 1,2-Bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, an intracellular Ca(2+) chelator, blocked early increases in Ca(2+) levels and inhibited beta-Lap-mediated mitochondrial membrane depolarization, intracellular ATP depletion, specific and unique substrate proteolysis, and apoptosis. The extracellular Ca(2+) chelator, EGTA, inhibited later apoptotic end points (observed >8 h, e.g. substrate proteolysis and DNA fragmentation), suggesting that later execution events were triggered by Ca(2+) influxes from the extracellular milieu. Collectively, these data suggest a critical, but not sole, role for Ca(2+) in the NQO1-dependent cell death pathway initiated by beta-Lap. Use of beta-Lap to trigger an apparently novel, calpain-like-mediated apoptotic cell death could be useful for breast and prostate cancer therapy.     

Low oxygen tension is a more potent promoter of chondrogenic differentiation than dynamic compression

During fracture healing and microfracture treatment of cartilage defects mesenchymal stem cells (MSCs) infiltrate the wound site, proliferate extensively and differentiate along a cartilaginous or an osteogenic lineage in response to local environmental cues. MSCs may be able to directly sense their mechanical environment or alternatively, the mechanical environment could act indirectly to regulate MSC differentiation by inhibiting angiogenesis and diminishing the supply of oxygen and other regulatory factors. Dynamic compression has been shown to regulate chondrogenesis of MSCs. In addition, previous studies have shown that a low oxygen environment promotes in vitro chondrogenesis of MSCs. The hypothesis of this study is that a low oxygen environment is a more potent promoter of chondrogenic differentiation of MSCs embedded in agarose hydrogels compared to dynamic compression. In MSC-seeded constructs supplemented with TGF-β3, GAG and collagen accumulation was higher in low oxygen conditions compared to normoxia. For normoxic and low oxygen culture GAG accumulation within the agarose hydrogel was inhomogeneous, with low levels of GAG measured in the annulus of constructs maintained in normoxic conditions. Dynamic compression did not significantly increase GAG or collagen accumulation in normoxia. However under low oxygen conditions, dynamic compression reduced GAG accumulation compared to free-swelling controls, but remained higher than comparable constructs maintained in normoxic conditions. This study demonstrates that continuous exposure to low oxygen tension is a more potent pro-chondrogenic stimulus than 1 h/day of dynamic compression for porcine MSCs embedded in agarose hydrogels.     

Synthetic hispidin, a PKC inhibitor, is more cytotoxic toward cancer cells than normal cells in vitro

The trypanocidal activity of naturally occurring 6-(3,4-dihydroxystyryl)-4-hydroxy-2-pyrone (hispidin) prompted us to examine its cytotoxic activity toward normal and cancerous cells in culture. Hispidin synthesized in our laboratory to a high degree of purity (checked by 1H and 13C NMR spectroscopy) was shown to be cytotoxic (between 10-3 mol/L and 10-7 mol/L) toward normal human MRC-5 fibroblasts, human cancerous keratinocytes (SCL-1 cell line), and human cancerous pancreatic duct cells (Capan-1 cell line). Interestingly, addition of hispidin in three successive doses (between 10-5 mol/L and 10-7 mol/L) led to a 100-fold increase in activity with an enhanced activity on cancer cells compared to normal cells (50%). Synthetic hispidin was found to inhibit isoform of protein kinase C (IC50 of 2 × 10-6 mol/L), but not E. coli and placental type XV alkaline phosphatases. The enhanced activity of hispidin toward the cancerous cell lines is discussed.     

Anosmin-1 involved in neuronal cell migration is hypoxia inducible and cancer regulated

Functional expression of KAL1 gene is critical in the migration of GnRH neurons from the olfactory placode to the hypothalamus in embryogenesis. This gene thus far has not been shown to play a functional role in any other physiological or pathological process either in the developed brain or in peripheral tissues. We show here that KAL1 gene expression is decreased in early stage and increased in later stages of cancers. Screening of colon, lung and ovarian cancer cDNA panels indicated significant decrease in KAL1 expression in comparison to corresponding uninvolved tissues. However, KAL1 expression increased with the progression of cancer from early (I and II) stages to later (III and IV) stages of the cancer. There was a direct correlation between the TGF-β and KAL1 expression in colon cancer cDNA. Using colon cancer cell lines, we showed that TGF-β induces KAL1 gene expression and secretion of anosmin-1 protein (KAL1 coded protein). We further report that hypoxia induces anosmin-1 expression; anosmin-1 protects cancer cells from apoptosis activated by hypoxia and increases cancer cell mobility. Using siRNA technique we found that KAL1 expression following hypoxia is hypoxia-inducible factor (HIF-1) α dependent. Our results suggest that KAL1 gene expression plays an important role in cancer metastasis and protection from apoptosis.     

MAPK signaling is involved in camptothecin-induced cell death

Camptothecin, a topoisomerase I inhibitor, is a well-known anticancer drug. However, its mechanism has not been well studied in human gastric cancer cell lines. Camptothecin induced apoptotic cell death in human gastric cancer cell line AGS. Z-VAD-fmk, pan-caspase inhibitor, blocked apoptotic phenotypes induced by camptothecin suggesting that caspases are involved in camptothecin-induced cell death. An inhibitor of caspase-6 or -8 or -9 did not prevent cell death by camptothecin. Various protease inhibitors failed to prevent camptothecin-induced cell death. These results suggest that only few caspases are involved in camptothecin-induced cell death. Camptothecin induced phosphorylation of ERK1/2, JNK, and p38 MAPK, in a dose and time-dependent manner in AGS. Z-VAD-fmk did not affect MAPK signaling induced by camptothecin suggesting that caspase signaling occurs downstream of MAPK signaling. Blocking of p38 MAPK, but not ERK1/2, resulted in partial inhibition of cell death and PARP cleavage by camptothecin in AGS. Taken together, MAPK signaling is associated with apoptotic cell death by camptothecin.     

Naringenin is a novel inhibitor of Dictyostelium cell proliferation and cell migration

Naringenin is a flavanone compound that alters critical cellular processes such as cell multiplication, glucose uptake, and mitochondrial activity. In this study, we used the social amoeba, Dictyostelium discoideum, as a model system for examining the cellular processes and signaling pathways affected by naringenin. We found that naringenin inhibited Dictyostelium cell division in a dose-dependent manner (IC(50) approximately 20 microM). Assays of Dictyostelium chemotaxis and multicellular development revealed that naringenin possesses a previously unrecognized ability to suppress amoeboid cell motility. We also found that naringenin, which is known to inhibit phosphatidylinositol 3-kinase activity, had no apparent effect on phosphatidylinositol 3,4,5-trisphosphate synthesis in live Dictyostelium cells; suggesting that this compound suppresses cell growth and migration via alternative signaling pathways. In another context, the discoveries described here highlight the value of using the Dictyostelium model system for identifying and characterizing the mechanisms by which naringenin, and related compounds, exert their effects on eukaryotic cells.