Kuang Yu Chen

Green tea epigallocatechin gallate shows a pronounced growth inhibitory effect on cancerous cells but not on their normal counterparts

(-)-Epigallocatechin gallate (EGCG), a catechin polyphenol compound, represents the main ingredient of green tea extract. Although EGCG has been shown to be growth inhibitory in a number of tumor cell lines, it is not clear whether the effect is cancer-specific. In this study we compared the effect of EGCG on the growth of SV40 virally transformed WI38 human fibroblasts (WI38VA) with that of normal WI38 cells. The IC50 value of EGCG was estimated to be 120 and 10 microM for WI38 and WI38VA cells, respectively. Thus, EGCG at 40 microM completely inhibited the growth of WI38VA cells, but had little or no inhibitory effect on the growth of WI38 cells. Similar differential growth inhibition was also observed between a human colorectal cancer cell line (Caco-2), a breast cancer cell line (Hs578T) and their respective normal counterparts. EGCG at a concentration range of 40-200 microM induced a significant amount of apoptosis in WI38VA cultures, but not in WI38 cultures, as determined by terminal deoxynucleotidyl transferase assay. After exposure to EGCG at 200 microM for 8 h, more than 50% of WI38VA cells in a confluent culture became apoptotic. In contrast, less than 1% of WI38 cells displayed apoptotic labeling under the same condition. EGCG did not affect the serum-induced expression of c-fos and c-myc genes in normal WI38 cells. However, it significantly enhanced their expression in transformed W138VA cells. It is possible that differential modulation of certain genes, such as c-fos and c-myc, may cause differential effects of EGCG on the growth and death of cancer cells.

Tandem affinity purification revealed the hypusine-dependent binding of eukaryotic initiation factor 5A to the translating 80S ribosomal complex

Eukaryotic initiation factor 5A (eIF5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post‐translationally in two steps by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genes encoding eIF5A or deoxyhypusine synthase are essential for cell survival and proliferation. To determine the physiological function of eIF5A, we have employed the tandem affinity purification (TAP) method and mass spectrometry to search for and identify the potential eIF5A‐interacting proteins. The TAP‐tag was fused in‐frame to chromosomal TIF51A gene and eIF5A‐TAP fusion protein expressed at its natural level was used as the bait to fish out its interacting partners. At salt concentrations of 150 mM, deoxyhypusine synthase was the only protein bound to eIF5A. As salt concentrations were lowered to 125 mM or less, eIF5A interacted with a set of proteins, which were identified as the components of the 80S ribosome complex. The eIF5A–ribosome interaction was sensitive to RNase and EDTA treatments, indicating the requirement of RNA and the joining of 40S and 60S ribosomal subunits for the interaction. Importantly, a single mutation of hypusine to arginine completely abolished the eIF5A–ribosome interaction. Sucrose gradient sedimentation analysis of log versus stationary phase cells and eIF3 mutant strain showed that the endogenous eIF5A co‐sedimented with the actively translating 80S ribosomes and polyribosomes in an RNase‐ and EDTA‐sensitive manner. Our study demonstrates for the first time that eIF5A interacts in a hypusine‐dependent manner with a molecular complex rather than a single protein, suggesting that the essential function of eIF5A is mostly likely mediated through its interaction with the actively translating ribosomes.

Identification of mRNA that binds to eukaryotic initiation factor 5A by affinity co-purification and differential display

Eukaryotic initiation factor 5A (eIF-5A) is the only protein in nature that contains hypusine, an unusual amino acid formed post-translationally by deoxyhypusine synthase and deoxyhypusine hydroxylase. Genetic and pharmacological evidence suggests that eIF-5A is essential for cell survival and proliferation. However, the precise function and interacting partners of eIF-5A remain unclear. We have shown previously that eIF-5A can bind to RRE (Rev-response element) and U6 RNA in vitro. Using SELEX (systematic evolution of ligands by exponential enrichment), we have also shown that eIF-5A is capable of binding to RNA in a sequence-specific manner [Xu and Chen (2001) J. Biol. Chem. 276, 2555-2561]. In the present paper, we show that the identification of mRNA species that bind to eIF-5A can be achieved by affinity co-purification and PCR differential display. Using this approach with three sets of anchoring and arbitrary primers, we have found 20 RNA sequences that co-purified specifically with eIF-5A. Five of them contained AAAUGU, the putative eIF-5A-interacting element that we identified previously using the SELEX method. Direct binding of the cloned RNA to eIF-5A could be demonstrated by electrophoretic mobility-shift assay. BLAST analysis revealed that the eIF-5A-interacting RNAs encode proteins such as ribosomal L35a, plasminogen activation inhibitor mRNA-binding protein, NADH dehydrogenase subunit and ADP-ribose pyrophosphatase. Some, however, encode hypothetical proteins. All the cloned RNAs have the potential to form extensive stem-loop structures.

Studies on the regulation of ornithine decar☐ylase activity by the microtubules: The effect of colchicine and vinblastine

Abstract Colchicine and vinblastine in micromolar concentrations inhibit the activity of ornithine decar☐ylase (E.C.4.1.1.17) (ODC), of mouse leukemia L1210 cells, which has been stimulated by dilution of the cells with fresh medium and serum. The colchicine analogues, lumicolchicine and colchiceine, which do not affect microtubular strcuture, do not inhibit ODC activity even at 10 −4 M. However, it appears that disruption of the microtubular structure is not in itself enough to inhibit ODC activity but that one or more additional temperature dependent steps are involved. We propose that the microtubule system is one of a series of components which regulates ODC activity.

Effects of inhibitors of deoxyhypusine synthase on the differentiation of mouse neuroblastoma and erythroleukemia cells

Deoxyhpusine synthase catalyzes the conversion of lysine to deoxyhypusine residue on the eukaryotic initiation factor 5A (eIF-5A) precursor using spermidine as the substrate. Subsequent hydroxylation of the deoxyhypusine residue completes hypusine formation on eIF-5A. Polyamines (putrescine, spermidine, and spermine) have been implicated in tumor growth and differentiation. Because deoxyhypusine/hypusine formation is one of the most specific polyamine-dependent biochemical events, we decided to use N1-guanyl-1,7-diaminoheptane (GC7), a potent inhibitor for deoxyhypusine synthase, to assess the role of hypusine formation on tumor growth and differentiation. GC7 suppressed the growth of N2a mouse neuroblastoma cells and DS19 murine erythroleukemia cells at micromolar concentrations. However, within a narrow concentration range, GC7 could promote the differentiation of mouse neuroblastoma cells in the presence of suboptimal amount of dibutyryl cAMP. In contrast, GC7 blocked the differentiation of DS19 cells induced with hexamethylene bisacetamide. Polyamine depletion by difluoromethyl ornithine (DFMO) has previously been shown to promote differentiation of neuroblastoma cells but inhibits erythrodifferentiation. Since our studies demonstrated that GC7 mimics the action of DFMO on tumor differentiation, it is likely that the effect of DFMO on tumor differentiation is mediated by hypusine formation and that GC7 represents a more specific inhibitor that can alter the differentiation program in certain tumor cells.

Subcellular localization of the hypusine‐containing eukaryotic initiation factor 5A by immunofluorescent staining and green fluorescent protein tagging

Eukaryotic initiation factor 5A (eIF‐5A) is the only protein in nature that contains hypusine, an unusual amino acid residue formed posttranslationally by deoxyhypusine synthase and deoxyhypusine hydroxylase. Although the eIF‐5A gene is essential for cell survival and proliferation, the precise function and localization of eIF‐5A remain unclear. In this study, we have determined the subcellular distribution of eIF‐5A by indirect immunofluorescent staining and by direct visualization of green fluorescent protein tagged eIF‐5A (GFP‐eIF5A). Immunofluorescent staining of the formaldehyde‐fixed cells showed that eIF‐5A was present in both the nucleus and cytoplasm. Only the nuclear eIF‐5A was resistant to Triton extraction. Direct visualization of GFP tagged eIF‐5A in living cells revealed the same whole‐cell distribution pattern. However, a fusion of an additional pyruvate kinase (PK) moiety into GFP‐eIF‐5A precluded the nuclear localization of GFP‐PK‐eIF‐5A fusion protein. Fusion of the GFP‐PK tag with three different domains of eIF‐5A also failed to reveal any nuclear localization of the fusion proteins, suggesting the absence of receptor‐mediated nuclear import. Using interspecies heterokaryon fusion assay, we could detect the nuclear export of GFP‐Rev, but not of GFP‐eIF‐5A. The whole‐cell distribution pattern of eIF‐5A was recalcitrant to the treatments that included energy depletion, heat shock, and inhibition of transcription, translation, polyamine synthesis, or CRM1‐dependent nuclear export. Collectively, our data indicate that eIF‐5A gains nuclear entry via passive diffusion, but it does not undergo active nucleocytoplasmic shuttling. J. Cell. Biochem. 86: 590–600, 2002.

The importance of natural product characterization in studies of their anti‐inflammatory activity

The knowledge that natural products provide a rich source for therapeutic discovery has led to the development of many of the worlds most commonly used drugs. In view of the growing need for effective anti-inflammatory agents, the potential for natural products to serve as safe and effective therapeutic agents has gained increasing attention. However, polymolecular extracts must be rigorously evaluated and chemically characterized to insure adequate consistency in performance. The research in this field has been plagued by inconsistencies due in part to inadequate chemical characterization and documentation, making comparison of results across studies very difficult. Analytical chemistry and molecular methods now exist to insure sufficient transparency to avoid this limitation. Further, our understanding of the complexity of inflammation has advanced to enable significant insight into the mechanism of action of these natural extracts. Here, we review the inflammatory pathways targeted by many therapeutic agents, discuss the value of natural products as anti-inflammatory agents, review approaches for their biological and chemical evaluation, and highlight challenges to the field. We present two examples highlighting the rigorous use of cell, molecular, and chemical methods for characterization and quality control as templates for future studies of anti-inflammatory activity of natural products.

Regulation of dihydrofolate reductase gene expression and E2F components in human diploid fibroblasts during growth and senescence.

The induction of dihydrofolate reductase (DHFR), a key enzyme in DNA biosynthesis that is induced just before the onset of S phase, is markedly attenuated in senescent human fibroblasts (Pang and Chen, 1994, J. Cell. Physiol., 160:531–538). Footprinting analysis of the 365 bp promoter region of the human DHFR gene (−381 to −17) indicated that nuclear proteins bind to a cluster of cis‐elements, including two overlapping E2F binding sequences, two Sp1 sites, and one Yi sequence. Gel mobility shift assays were performed to assess the role of each cis‐element in the regulation of DHFR gene expression. We found that (1) Sp1 binding activity was constitutively expressed throughout the cell cycle in early passage and senescent cells; (2) Yi binding activity was undetectable in both early passage and senescent cells; and (3) E2F binding activity was serum‐inducible, senescence‐dependent, and prominent in presenescent cells but strikingly diminished in senescent cells. Northern blot analysis of the expression of E2F and DP family members showed that the E2F‐1, E2F‐4, and E2F‐5 mRNA was growth‐ and senescence‐dependent, whereas E2F‐3, DP‐1, and DP‐2 expression was constitutive and senescence‐independent. In contrast, E2F‐2 mRNA was not detectable in IMR‐90 or WI‐38 human fibroblasts. Western blot analysis showed that among the E2F‐associated proteins, the expression of E2F‐1, cyclin A, and cyclin B but not p107 was cell cycle‐ and senescence‐dependent. A nuclear extract mixing experiment suggested that an inhibitory factor may further reduce E2F binding activity in senescent cells.

Effects of the black tea polyphenol theaflavin-2 on apoptotic and inflammatory pathways in vitro and in vivo

SCOPE Theaflavin-2 (TF-2), a major component of black tea extract, induces apoptosis of human colon cancer cells and suppresses serum-induced cyclooxygenase-2 (COX-2) expression 1. Here, we explored the mechanisms for activation of apoptosis, evaluated the impact on inflammatory genes in a broader panel of cells and tested whether topical anti-inflammatory effects could be observed in vivo. METHODS AND RESULTS TF-2 triggered apoptosis in five other transformed cancer cell lines, inducing cell shrinkage, membrane blebbing, and mitochondrial clustering within 3 h of treatment. Among a set of pro-apoptotic genes, TF-2 quickly induced the up-regulation of P53 and BAX, suggesting mitochondria as the primary target. Using a cell model for inflammatory response, we showed that TF-2 suppressed the 12-O-tetradecanoylphorbol-13-acetate-induced COX-2 gene expression, and also down-regulated TNF-α, iNOS, ICAM-1, and NFκB. A reporter gene assay showed that TF-2 down-regulated COX-2 at the transcriptional level. We also demonstrated that TF-2 exhibited anti-inflammatory activity in two mouse models of inflammation. Topical application with TF-2 significantly reduced ear edema and produced a pattern of gene down-regulation similar to that observed in the cell model. CONCLUSION These results suggest that the anti-inflammatory and pro-apoptotic activity of TF-2 may be exploited therapeutically in cancer and other diseases associated with inflammation.

Riluzole Increases the Amount of Latent HSF1 for an Amplified Heat Shock Response and Cytoprotection

Background Induction of the heat shock response (HSR) and increased expression of the heat shock proteins (HSPs) provide mechanisms to ensure proper protein folding, trafficking, and disposition. The importance of HSPs is underscored by the understanding that protein mis-folding and aggregation contribute centrally to the pathogenesis of neurodegenerative diseases. Methodology/Principal Findings We used a cell-based hsp70-luciferease reporter gene assay system to identify agents that modulate the HSR and show here that clinically relevant concentrations of the FDA-approved ALS drug riluzole significantly increased the heat shock induction of hsp70-luciferse reporter gene. Immuno-Western and -cytochemical analysis of HSF1 show that riluzole increased the amount of cytosolic HSF1 to afford a greater activation of HSF1 upon heat shock. The increased HSF1 contributed centrally to the cytoprotective activity of riluzole as hsf1 gene knockout negated the synergistic activity of riluzole and conditioning heat shock to confer cell survival under oxidative stress. Evidence of a post-transcriptional mechanism for the increase in HSF1 include: quantitation of mRNAhsf1 by RT-PCR showed no effect of either heat shock or riluzole treatment; riluzole also increased the expression of HSF1 from a CMV-promoter; analysis of the turnover of HSF1 by pulse chase and immunoprecipitation show that riluzole slowed the decay of [35S]labeled-HSF1. The effect of riluzole on HSF1 was qualitatively different from that of MG132 and chloroquine, inhibitors of the proteasome and lysosome, respectively, and appeared to involve the chaperone-mediated autophagy pathway as RNAi-mediated knockdown of CMA negated its effect. Conclusion/Significance We show that riluzole increased the amount of HSF1 to amplify the HSR for cytoprotection. Our study provides novel insight into the mechanism that regulates HSF1 turnover, and identifies the degradation of HSF1 as a target for therapeutics intervention.