Jong-Soo Chang

The phytoestrogen genistein induces thymic and immune changes: A human health concern?

Use of soy-based infant formulas and soy/isoflavone supplements has aroused concern because of potential estrogenic effects of the soy isoflavones genistein and daidzein. Here we show that s.c. genistein injections in ovariectomized adult mice produced dose-responsive decreases in thymic weight of up to 80%. Genisteins thymic effects occurred through both estrogen receptor (ER) and non-ER-mediated mechanisms, as the genistein effects on thymus were only partially blocked by the ER antagonist ICI 182,780. Genistein decreased thymocyte numbers up to 86% and doubled apoptosis, indicating that the mechanism of the genistein effect on loss of thymocytes is caused in part by increased apoptosis. Genistein injection caused decreases in relative percentages of thymic CD4+CD8− and double-positive CD4+CD8+ thymocytes, providing evidence that genistein may affect early thymocyte maturation and the maturation of the CD4+CD8− helper T cell lineage. Decreases in the relative percentages of CD4+CD8− thymocytes were accompanied by decreases in relative percentages of splenic CD4+CD8− cells and a systemic lymphocytopenia. In addition, genistein produced suppression of humoral immunity. Genistein injected at 8 mg/kg per day produced serum genistein levels comparable to those reported in soy-fed human infants, and this dose caused significant thymic and immune changes in mice. Critically, dietary genistein at concentrations that produced serum genistein levels substantially less than those in soy-fed infants produced marked thymic atrophy. These results raise the possibility that serum genistein concentrations found in soy-fed infants may be capable of producing thymic and immune abnormalities, as suggested by previous reports of immune impairments in soy-fed human infants.

Resveratrol inhibits phorbol myristate acetate-induced matrix metalloproteinase-9 expression by inhibiting JNK and PKC δ signal transduction

Proteolytic degradation of the extracellular matrix and tumor metastasis correlate with the expression of endopeptidases known as matrix metalloproteinases (MMPs). The expression of MMPs is regulated by cytokines and signal transduction pathways, including those activated by phorbol myristate acetate (PMA). We found that resveratrol, a phytoalexin present in grapes, significantly inhibits the PMA-induced increase in MMP-9 expression and activity. These effects of resveratrol are dose dependent and correlate with the suppression of MMP-9 mRNA expression levels. PMA caused about a 23-fold increase in MMP-9 promoter activity, which was suppressed by resveratrol. Transient transfection utilizing MMP-9 constructs, in which specific transcriptional factors were mutagenized, indicated that the effects of PMA and resveratrol were mediated via an activator protein-1 and nuclear factor-κB response element. Resveratrol inhibited PMA-mediated activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)-δ activation. Therefore, we conclude that the MMP-9 inhibition activity of resveratrol and its inhibition of JNK and PKC-δ may have a therapeutic potential, given that a novel means of controlling growth and invasiveness of tumors.

Pleckstrin Homology Domains of Phospholipase C-γ1 Directly Interact with β-Tubulin for Activation of Phospholipase C-γ1 and Reciprocal Modulation of β-Tubulin Function in Microtubule Assembly

Phosphoinositide-specific phospholipase C-γ1 (PLC-γ1) has two pleckstrin homology (PH) domains, an N-terminal domain and a split PH domain. Here we show that pull down of NIH3T3 cell extracts with PLC-γ1 PH domain-glutathione S-transferase fusion proteins, followed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry, identified β-tubulin as a binding protein of both PLC-γ1 PH domains. Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of α- and β-tubulin heterodimers in all eukaryotic cells. PLC-γ1 and β-tubulin colocalized in the perinuclear region in COS-7 cells and cotranslocated to the plasma membrane upon agonist stimulation. Membrane-targeted translocation of depolymerized tubulin by agonist stimulation was also supported by immunoprecipitation analyses. The phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolyzing activity of PLC-γ1 was substantially increased in the presence of purified tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that β-tubulin activates PLC-γ1. Furthermore, indirect immunofluorescent microscopy showed that PLC-γ1 was highly concentrated in mitotic spindle fibers, suggesting that PLC-γ1 is involved in spindle fiber formation. The effect of PLC-γ1 in microtubule formation was assessed by overexpression and silencing PLC-γ1 in COS-7 cells, which resulted in altered microtubule dynamics in vivo. Cells overexpressing PLC-γ1 showed higher microtubule densities than controls, whereas PLC-γ1 silencing with small interfering RNAs led to decreased microtubule network densities as compared with control cells. Taken together, our results suggest that PLC-γ1 and β-tubulin transmodulate each other, i.e. that PLC-γ1 modulates microtubule assembly by β-tubulin, and β-tubulin promotes PLC-γ1 activity.

Pleckstrin homology domain of phospholipase C-γ1 directly binds to 68-kDa neurofilament light chain

Phosphoinositide-specific phospholipase C-γ1 (PLC-γ1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-γ1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-γ1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-γ1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-γ1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-γ1. Our results suggest that PLC-γ1-associated NF-L sequesters the phospholipid from the PH domain of PLC-γ1.

The colchicine derivative CT20126 shows a novel microtubule-modulating activity with apoptosis

New colchicine analogs have been synthesized with the aim of developing stronger potential anticancer activities. Among the analogs, CT20126 has been previously reported to show immunosuppressive activities. Here, we report that CT20126 also shows potential anticancer effects via an unusual mechanism: the modulation of microtubule integrity and cell cycle arrest at the G2/M phase before apoptosis. When we treated COS-7 cells with CT20126 (5 μM), the normal thread-like microtubules were disrupted into tubulin dimers within 10 min and thereafter repolymerized into short, thick filaments. In contrast, cells treated with the same concentration of colchicine exhibited microtubule depolymerization after 20 min and never underwent repolymerization. Furthermore, optical density (OD) analysis (350 nm) with purified tubulin showed that CT20126 had a higher repolymerizing activity than that of Taxol, a potent microtubule-polymerizing agent. These results suggest that the effects of CT20126 on microtubule integrity differ from those of colchicine: the analog first destabilizes microtubules and then stabilizes the disrupted tubulins into short, thick polymers. Furthermore, CT20126 induced a greater level of apoptotic activity in Jurkat T cells than colchicine (assessed by G2/M arrest, caspase-3 activation and cell sorting). At 20 nM, CT20126 induced 47% apoptosis among Jurkat T cells, whereas colchicine induced only 33% apoptosis. Our results suggest that the colchicine analog CT20126 can potently induce apoptosis by disrupting microtubule integrity in a manner that differs from that of colchicine or Taxol.

A double point mutation in PCL-γ1 (Y509A/F510A) enhances Y783 phosphorylation and inositol phospholipid-hydrolyzing activity upon EGF stimulation

Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-γ1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-γ1, abolished interactions with translational elongation factor 1-α. Here, we report that the Y509A/F510A mutant PLC-γ1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-γ1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-γ1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-γ1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-γ1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-γ1 activation in vivo.

Phospholipase C-γ1 Activation by Direct Interaction with β-Tubulin Isotypes

Phosphoinositide-specific phospholipase C-γ1 (PLC-γ1) has pivotal roles in cellular signaling by producing second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG). Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of α- and β-tubulin heterodimers in all eukaryotic cells. In humans, six β-tubulin isotypes have been identified which display a distinct pattern of tissue expression. Previously we found that PLC-γ1 and one of four β-tubulin isotypes including β1, β2, β3 and β6, colocalized in COS-7 cells and cotranslocated to the plasma membrane to activate PLC-γ1 upon agonist stimulation. In the present study, we demonstrate that the remaining two, tubulin β4 and β5, also showed a potential to activate PLC-γ1. The phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolyzing activity of PLC-γ1 was substantially increased in the presence of purified β4 and β5 tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that tubulin β4 and β5 also activate PLC-γ1. Taken together, our results suggest that all the β-tubulin isotype activates PLC-γ1 activity to regulate cellular signaling.

Corosolic Acid Induces Non-Apoptotic Cell Death through Generation of Lipid Reactive Oxygen Species Production in Human Renal Carcinoma Caki Cells

Corosolic acid is one of the pentacyclic triterpenoids isolated from Lagerstroemia speciose and has been reported to exhibit anti-cancer and anti-proliferative activities in various cancer cells. In the present study, we investigated the molecular mechanisms of corosolic acid in cancer cell death. Corosolic acid induces a decrease of cell viability and an increase of cell cytotoxicity in human renal carcinoma Caki cells. Corosolic acid-induced cell death is not inhibited by apoptosis inhibitor (z-VAD-fmk, a pan-caspase inhibitor), necroptosis inhibitor (necrostatin-1), or ferroptosis inhibitors (ferrostatin-1 and deferoxamine (DFO)). Furthermore, corosolic acid significantly induces reactive oxygen species (ROS) levels, but antioxidants (N-acetyl-l-cysteine (NAC) and trolox) do not inhibit corosolic acid-induced cell death. Interestingly, corosolic acid induces lipid oxidation, and α-tocopherol markedly prevents corosolic acid-induced lipid peroxidation and cell death. Anti-chemotherapeutic effects of α-tocopherol are dependent on inhibition of lipid oxidation rather than inhibition of ROS production. In addition, corosolic acid induces non-apoptotic cell death in other renal cancer (ACHN and A498), breast cancer (MDA-MB231), and hepatocellular carcinoma (SK-Hep1 and Huh7) cells, and α-tocopherol markedly inhibits corosolic acid-induced cell death. Therefore, our results suggest that corosolic acid induces non-apoptotic cell death in cancer cells through the increase of lipid peroxidation.

Sphingobacterium sp. SW-09 Effectively Degrades Phenanthrene, a Polycyclic Aromatic Hydrocarbon, in a Soil Microcosm

We isolated a potent phenanthrene-degrading bacterium from oil-contaminated soils of Suzhou, China, and assessed the potential use of these bacteria for bioremediation of soils contaminated by polycyclic aromatic hydrocarbons (PAHs) in a microcosm. Based on 16S rDNA sequencing, we identified this bacteria as Sphigobacterium sp. SW-09. By PCR amplification, we also identified catechol 2,3-dioxygenase genes (nahH genes) mediating PAH degradation. Staphylococcus sp. KW-07, which has been identified in our previous study, showed potential for use in bioremediation of oil-contaminated soils. In this experiment, we compared the rate of phenanthrene-degradation between Staphylococcus sp. KW-07 and Sphingobacterium sp. SW-09 in a microcosm condition. Newly isolated Sphingobacterium sp. SW-09 showed a higher phenanthrene-degradation rate than that of Staphylococcus sp. KW-07 in soil microcosms. Together, our results suggest that the Sphingobacterim sp. SW-09 strain isolated from the Suzhou area may also be useful in bioremediation of PAH-contaminated soils.