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.

Effect of microbial inoculants on fermentation quality and aerobic stability of sweet potato vine silage.

OBJECTIVE: This study was conducted to evaluate the effect of homo or hetero fermentative inoculants on fermentation quality and aerobic stability of sweet potato vine (SPV) silage containing Italian ryegrass hay as moisture absorbent. METHODS: The SPV was harvested at 15% dry matter, mixed with Italian ryegrass hay at 1:1 ratio on a fresh weight basis, and chopped to 3 to 5 cm length. After then, the chopped forage mixture was ensiled into 20-L mini silos in quadruplicate for 7, 48, and 100 days after application of microbial inoculants at 1.2×105 colony forming units (cfu)/g of forage following: no inoculant (CON), Lactobacillus plantarum as a homo fermentative (LP), Lactobacillus buchneri as a hetero fermentative (LB), and mixture of LP and LB at 1:1 ratio as a combo fermentative (MIX). RESULTS: The LP and MIX silages had lowest pH (p<0.001) on 7 and 48 days, while MIX and CON silages had greatest lactate concentrations (p<0.05) on 7 and 48 days, respectively. Acetate concentrations were highest (p<0.01) in LB and MIX silages on 7 days, and in LB silage on 48 days, while lactate to acetate ratios were lowest (p<0.001) in LB silages. The chemical compositions and nutrient digestibility of silage ensiled for 100 days was not affected by inoculants. On 100 days of ensiling, LB silage had lowest (p<0.01) lactate concentration and lactate to acetate ratio, but highest acetate concentration. Aerobic stability was highest (p<0.001) in LB silage followed in MIX silage. On contrast, LB silage had lowest (p<0.05) lactic acid bacteria and mold. CONCLUSION: The results indicated that application of LB solely had a better effect on aerobic stability than not only LP, but also MIX. However, LP application did not show beneficial effects from the viewpoints of fermentation quality and aerobic stability compared to CON.

Phospholipase Cgamma1 negatively regulates growth hormone signalling by forming a ternary complex with Jak2 and protein tyrosine phosphatase-1B.

Growth hormone binds to its membrane receptor (GHR), whereby it regulates many cellular functions, including proliferation, differentiation and chemotaxis. However, although the activation of growth hormone-mediated signalling is well understood, the precise mechanism responsible for its regulation has not been elucidated. Here, we demonstrate that phospholipase Cgamma1 (PLCgamma1) modulates the action of growth hormone-mediated signalling by interacting with tyrosine kinase Jak2 (janus kinase 2) in a growth hormone-dependent manner. In the absence of PLCgamma1 (PLCgamma1(-/-)), growth hormone-induced JAK2 and STAT5 phosphorylation significantly increased in mouse embryonic fibroblasts (MEFs). Furthermore, the re-expression of PLCgamma1 reduced growth hormone-induced Jak2 activation. Growth hormone-induced Jak2 phosphorylation was enhanced by siRNA-specific knockdown of PLCgamma1. Interestingly, PLCgamma1 physically linked Jak2 and protein tyrosine phosphatase-1B (PTP-1B) by binding to both using different domains, and this process was implicated in the modulation of cytokine signalling through Jak2. In addition, in PLCgamma1(-/-) MEFs, growth hormone-dependent c-Fos activation was upregulated and growth hormone-induced proliferation was potentiated. These results suggest that PLCgamma1 has a key function in the regulation of growth hormone-mediated signalling by negatively regulating Jak2 activation.

The insect peptide CopA3 inhibits lipopolysaccharide-induced macrophage activation.

We recently demonstrated that the insect peptide CopA3 (LLCIALRKK), a disulfide-linked dimeric peptide, exerts antimicrobial and anti-inflammatory activities in a mouse colitis model. Here, we examined whether CopA3 inhibited activation of macrophages by LPS. Exposure of an unseparated mouse peritoneal cell population or isolated peritoneal macrophages to LPS markedly increased secretion of IL-6 and TNF-α; these effects were significantly inhibited by CopA3 treatment. The inhibitory effect of CopA3 was also evident in murine macrophage cell line, RAW 264.7. Western blotting revealed that LPS-induced activation of STAT1 and STAT5 in macrophages was significantly inhibited by CopA3. Inhibition of JAK (STAT1/STAT5 kinase) with AG490 markedly reduced the production of IL-6 and TNF-α in macrophages. Collectively, these observations suggest that CopA3 inhibits macrophage activation by inhibiting activating phosphorylations of the transcription factors, STAT1 and STAT5, and blocking subsequent production of IL-6 and TNF-α and indicate that CopA3 may be useful as an immune-modulating agent.

Clostridium difficile toxin A decreases acetylation of tubulin, leading to microtubule depolymerization through activation of histone deacetylase 6, and this mediates acute inflammation.

Clostridium difficile toxin A is known to cause actin disaggregation through the enzymatic inactivation of intracellular Rho proteins. Based on the rapid and severe cell rounding of toxin A-exposed cells, we speculated that toxin A may be involved in post-translational modification of tubulin, leading to microtubule instability. In the current study, we observed that toxin A strongly reduced α-tubulin acetylation in human colonocytes and mouse intestine. Fractionation analysis demonstrated that toxin A-induced α-tubulin deacetylation yielded monomeric tubulin, indicating the presence of microtubule depolymerization. Inhibition of the glucosyltransferase activity against Rho proteins of toxin A by UDP-2',3'-dialdehyde significantly abrogated toxin A-induced α-tubulin deacetylation. In colonocytes treated with trichostatin A (TSA), an inhibitor of the HDAC6 tubulin deacetylase, toxin A-induced α-tubulin deacetylation and loss of tight junction were completely blocked. Administration of TSA also attenuated proinflammatory cytokine production, mucosal damage, and epithelial cell apoptosis in mouse intestine exposed to toxin A. These results suggest that toxin A causes microtubule depolymerization by activation of HDAC6-mediated tubulin deacetylation. Indeed, blockage of HDAC6 by TSA markedly attenuates α-tubulin deacetylation, proinflammatory cytokine production, and mucosal damage in a toxin A-induced mouse enteritis model. Tubulin deacetylation is an important component of the intestinal inflammatory cascade following toxin A-mediated Rho inactivation in vitro and in vivo.

Upregulation of DR5 and Downregulation of Survivin by IITZ-01, Lysosomotropic Autophagy Inhibitor, Potentiates TRAIL-Mediated Apoptosis in Renal Cancer Cells via Ubiquitin-Proteasome Pathway.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively is able to increase apoptosis in cancer cells as agent with minimum toxicity to noncancerous cells. However, all cancer cells are not sensitive to TRAIL-induced apoptosis. In this study, we showed the sub-lethal concentrations of a lysosomotropic autophagy inhibitor, IITZ-01, sensitizes cancer cells (renal, lung, and breast carcinoma) to TRAIL-induced apoptosis through DR5 upregulation and survivin downregulation through ubiquitin-proteasome pathway. Knockdown of DR5 or overexpression of survivin inhibited combined treatment with IITZ-01 and TRAIL-induced apoptosis. IITZ-01 downregulated protein expression of Cbl, ubiquitin E3 ligase, and decreased expression level of Cbl markedly led to increase DR5 protein expression and TRAIL sensitivity. Moreover, IITZ-01 decreased expression level of survivin protein via downregulation of deubiquitinase ubiquitin-specific protease 9X (USP9X) expression. Taken together, these results provide the first evidence that IITZ-01 enhances TRAIL-mediated apoptosis through DR5 stabilization by downregulation of Cbl and USP9X-dependent survivin ubiquitination and degradation in renal carcinoma cells.

The coffee diterpene kahweol induces apoptosis in human leukemia U937 cells through down-regulation of Akt phosphorylation and activation of JNK.

Kahweol, the coffee-specific diterpene, has been reported for its tumor cell growth inhibitory activity and anti-carcinogenic activity. The mechanism by which kahweol initiates apoptosis remains poorly understood. In the present study, we investigated the effect of kahweol on the apoptotic pathway in U937 human promonocytic cells. We show that kahweol induces apoptosis in association with the activation of caspase 3 and cytochrome c release from the mitochondria to the cytosol, as well as down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1 and XIAP). Kahweol altered the phosphorylation state of members of the MAPKs and Akt. Ectopic expression of Bcl-2 or constitutive active Akt (myr-Akt) in U937 cells attenuates kahweol-induced apoptosis. In addition, we have also shown that JNK and Akt signal pathway plays a crucial role in kahweol-induced apoptosis in U937 cells. Taken together, our results show the activity of kahweol to modulate multiple components in apoptotic response of human leukemia cells and raise the possibility a novel therapeutic strategy in hematological malignancies.

Phospholipase D prevents etoposide-induced apoptosis by inhibiting the expression of early growth response-1 and phosphatase and tensin homologue deleted on chromosome 10.

Phospholipase D (PLD) has emerged as a critical regulator of cell proliferation and survival signaling. We show for the first time that elevated expression of PLD isozymes attenuates expression of the tumor suppressors early growth response-1 (Egr-1) and the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumor suppressor and apoptosis during etoposide treatment. When formation of phosphatidic acid was inhibited by overexpression of catalytically inactive PLD during etoposide treatment, expression of Egr-1 and PTEN and the apoptotic effect of etoposide were not inhibited. This suggests that PLD inhibits expression of these tumor suppressors and inhibits apoptosis. Deletion of a specific Egr-1-binding site present in the PTEN promoter blocked etoposide-induced PTEN activity and elevated expression of PLD decreased the sensitivity to apoptosis induced by ectopic expression of Egr-1. Etoposide-induced activation of Akt was potentiated by overexpression of PLD and PLD-stimulated suppression of Egr-1 was blocked by inhibition of phosphatidylinositol 3-kinase/Akt survival pathway at the both transcriptional and posttranscriptional levels. These results show that survival signals generated by PLD attenuate expression of Egr-1 by activation of phosphatidylinositol 3-kinase signaling pathway and induction of PTEN by Egr-1, which confers resistance to apoptosis.

RASAL3 preferentially stimulates GTP hydrolysis of the Rho family small GTPase Rac2.

Members of the Ras superfamily of small G-proteins serve as molecular switches of intracellular signaling pathways. Rac2 is a Rho subfamily GTPase switch that is specifically expressed in hematopoietic cells and regulates AKT activation in cell signaling. Ras activating protein-like 3 (RASAL3) is the recently identified Ras GTPase activating protein (GAP) that is also specifically expressed in hematopoietic cells and stimulates p21ras GTPase activity. The restricted expression of both Rac2 and RASAL3 suggests that they may serve critical roles in hematopoietic cell signaling. Here in the present study demonstrates that the catalytic domain of RASAL3 may also be able to interact with Rac2 and stimulate its GTPase activity in vitro. By contrast, p50 rhoGAP molecules did not markedly affect Rac2 GTPase activity, but did accelerate the activity of other Rho GTPases, including Rac1, RhoA and Cdc42. Collectively, the present results indicate, seemingly for the first time, that GAP activity for Rac2 is regulated by the RasGAP family protein, RASAL3.

In vitro assay of neurofilament light chain self-assembly using truncated mutants.

Neurofilaments (NFs) are heteropolymers composed of light (NF-L), middle (NF-M), and heavy (NF-H) subunits, present in most neurons. NF-L polymerizes on its own to provide a scaffold on which regular NFs form via the cross-bridging of NF-M or NF-H. To clarify the mechanism of regulation of NF-L self-assembly, we developed an assay using truncated mutant NF-L fused to glutathione-S transferase (GST). Western immunoblotting data show that the GST-fused head-rod domains of NF-L are necessary and sufficient for detecting assembled NF-L. The levels of self-assembled NF-L subunits detected using GST fusion proteins were consistent with those detected by electron microscopy and turbidity assay. Our results collectively imply that GST-fused head-rod domains of NF-L are critical tools for analyzing NF-L self-assembly in vitro.

Transmodulation between phospholipase D and c-Src enhances cell proliferation.

Phospholipase D (PLD) has been implicated in the signal transduction pathways initiated by several mitogenic protein tyrosine kinases. We demonstrate for the first time that most notably PLD2 and to a lesser extent the PLD1 isoform are tyrosine phosphorylated by c-Src tyrosine kinase via direct association. Moreover, epidermal growth factor induced tyrosine phosphorylation of PLD2 and its interaction with c-Src in A431 cells. Interaction between these proteins is via the pleckstrin homology domain of PLD2 and the catalytic domain of c-Src. Coexpression of PLD1 or PLD2 with c-Src synergistically enhances cellular proliferation compared with expression of either molecule. While PLD activity as a lipid-hydrolyzing enzyme is not affected by c-Src, wild-type PLDs but not catalytically inactive PLD mutants significantly increase c-Src kinase activity, up-regulating c-Src-mediated paxillin phosphorylation and extracellular signal-regulated kinase activity. These results demonstrate the critical role of PLD catalytic activity in the stimulation of Src signaling. In conclusion, we provide the first evidence that c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src. This transmodulation between c-Src and PLD may contribute to the promotion of cellular proliferation via amplification of mitogenic signaling pathways.

Pleckstrin homology domain of phospholipase C-gamma1 directly binds to 68-kDa neurofilament light chain.

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) 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-gamma1 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-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 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)P(2)] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.

Satellite cells isolated from adult Hanwoo muscle can proliferate and differentiate into myoblasts and adipose-like cells.

This study examined whether adult bovine muscle satellite cells from 30-month-old Hanwoo cattle are multipotential. The satellite cells were found to have the potential to proliferate and differentiate into myoblasts with the formation of multinucleated cells. In addition, treatment with the peroxisome proliferator activating receptor-gamma (PPARgamma) agonist, rosiglitazone, promoted their trans-differentiation into adipocytes with significant increases in glycerol accumulation and glycerol-3-phosphate dehydrogenase activity. Western blot analysis revealed that increased levels of the adipocyte fatty acid-binding protein, PPARgamma and of CCAAT/enhancer-binding protein were closely related to rosiglitazone-induced differentiation of the cells. These findings demonstrate that satellite cells from adult Hanwoo cattle are multipotent, and that their trans-differentiation into adipocytes can be induced by rosiglitazone.

Resveratrol inhibits phorbol myristate acetate-induced matrix metalloproteinase-9 expression by inhibiting JNK and PKC delta 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-kappaB response element. Resveratrol inhibited PMA-mediated activation of c-Jun N-terminal kinase (JNK) and protein kinase C (PKC)-delta activation. Therefore, we conclude that the MMP-9 inhibition activity of resveratrol and its inhibition of JNK and PKC-delta may have a therapeutic potential, given that a novel means of controlling growth and invasiveness of tumors.

Transcriptional and post-transcriptional regulation of the PKC delta gene by etoposide in L1210 murine leukemia cells: implication of PKC delta autoregulation.

Protein kinase C delta (PKC delta) plays an important role in the regulation of apoptosis in response to diverse anticancer agents. PKC delta is cleaved irreversibly to a catalytically active fragment in response to apoptotic stimuli; however, little information is available about the regulation of PKC delta gene expression. In this study, we found that the amount of steady-state PKC delta mRNA and protein was increased by etoposide in mouse L1210 leukemia cells. The transcriptional rate of the PKC delta gene and the stability of PKC delta mRNA were increased by treatment with etoposide, resulting in the accumulation of PKC delta protein. Rottlerin inhibited etoposide-induced PKC delta gene expression significantly, while Go6976, LY294002 and PD98059 had no effect. Further, both stable and adenovirus-mediated expression of a dominant negative PKC delta(KR) abrogated etoposide-induced PKC delta expression. Etoposide-stimulated PKC delta transcription but not PKC delta mRNA stability was blocked completely by pretreatment with rottlerin. Our data reveal a novel mechanism whereby PKC delta gene is regulated at the transcriptional and post-transcriptional level in the L1210 leukemia cell line.

Negative regulatory role of overexpression of PLC gamma 1 in the expression of early growth response 1 gene in rat 3Y1 fibroblasts.

The early growth response 1 (Egr-1) gene product is a transcription factor that functions as an oikis factor. Loss of Egr-1 expression is closely associated with tumor formation. Phospholipase Cgamma1 (PLCgamma1) is overexpressed in some tumors, and its overexpression causes anchorage-independent growth. Here we report that overexpression of PLCgamma1 and SH2-SH3 domain of PLCgamma1 decreased induction of Egr-1 and the Egr-1-regulated genes TSP-1 and PAI-1. Results from the nuclear run-on assay and transfection experiment with the proximal 455 base pair region of the Egr-1 promoter (-454 to +1) showed that Egr-1 transcriptional activity was suppressed in PLCgamma1-3Y1 cells whereas decay of Egr-1 mRNA was similar in both cell lines. Serum response element- and ternary complex factor Elk-1-mediated transcriptional activation of the reporter gene in response to EGF were also inhibited in PLCgamma1-3Y1 cells. Pretreatment with the protein synthesis inhibitor cycloheximide (CHX) partially abrogated the serum-induced suppression of Egr-1 transcription in PLCgamma1-3Y1 cells, suggesting that a CHX-sensitive factor(s) is involved in the suppression of Egr-1 transcription in PLCgamma1-3Y1 cells. Our results demonstrated that overexpression of PLCgamma1 functions as a negative modulator of the tumor suppressor Egr-1 gene expression, possibly through inhibition of Elk-1-dependent transcriptional activity.

A Simple Evaporation Method for Large-Scale Production of Liquid Crystalline Lipid Nanoparticles with Various Internal Structures.

We present a simple and industrially accessible method of producing liquid crystalline lipid nanoparticles with various internal structures based on phytantriol, Pluronic F127, and vitamin E acetate. Bilayer vesicles were produced when an ethanolic solution dissolving the lipid components was mixed with deionized water. After the evaporation of ethanol from the aqueous mixture, vesicles were transformed into lipid-filled liquid crystalline nanoparticles with well-defined internal structures such as hexagonal lattices (mostly inverted cubic Pn3m), lined or coiled pattern (inverted hexagonal H2), and disordered structure (inverse microemulsion, L2), depending on the compositions. Further studies suggested that their internal structures were also affected by temperature. The internal structures were characterized from cryo-TEM and small-angle X-ray scattering results. Microcalorimetry studies were performed to investigate the degree of molecular ordering/crystallinity of lipid components within the nanostructures. From the comparative studies, we demonstrated the present method could produce the lipid nanoparticles with similar characteristics to those made from a conventional method. More importantly, the production only requires simple tools for mixing and ethanol evaporation and it is possible to produce 10 kg or so per batch of aqueous lipid nanoparticles dispersions, enabling the large-scale production of the liquid crystalline nanoparticles for various biomedical applications.

Point mutations in the split PLC-gamma1 PH domain modulate phosphoinositide binding.

A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal (PH(1)) and a split PH domain (nPH(2) and cPH(2)). We previously reported that the split PH domain of PLC-gamma1 binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)P(2), we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-gamma1 nPH(2) domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-gamma1 nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-gamma1 molecules showed reduced PI(4,5)P(2) hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both PH(1) and nPH(2) domains are responsible for membrane-targeted translocation of PLC-gamma1 upon serum stimulation. Together, our data reveal that the amino acid residues Pro(500) and His(503) are critical for binding of PLC-gamma1 to one of its substrates, PI(4,5)P(2) in the membrane.

FMRFamide-expressing efferent neurons in eighth abdominal ganglion innervate hindgut in the silkworm, Bombyx mori.

The tetrapeptide FMRFamide is known to affect both neural function and gut contraction in a wide variety of invertebrates and vertebrates, including insect species. This study aimed to find a pattern of innervation of specific FMRFamide-labeled neurons from the abdominal ganglia to the hindgut of the silkworm Bombyx mori using the immunocytochemical method. In the 1st to the 7th abdominal ganglia, labeled efferent neurons that would innervate the hindgut could not be found. However, in the 8th abdominal ganglion, three pairs of labeled specific efferent neurons projected axons into the central neuropil to eventually innervate the hindgut. Both axons of two pairs of labeled cell bodies in the lateral rind and axons of one pair of labeled cell bodies in the posterior rind extended to the central neuropil and formed contralateral tracts of a labeled neural tract with a semi-circular shape. These labeled axons ran out to one pair of bilateral cercal nerves that extended out from the posterior end of the 8th abdominal ganglion and finally to the innervated hindgut. These results provide valuable information for detecting the novel function of FMRFamide-related peptides in metamorphic insect species.

Silibinin induces apoptosis of HT29 colon carcinoma cells through early growth response-1 (EGR-1)-mediated non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) up-regulation.

Silibinin, an effective anti-cancer and chemopreventive agent, has been shown to exert multiple effects on cancer cells, including inhibition of both cell proliferation and migration. However, the molecular mechanisms responsible for these effects are not fully understood. We observed that silibinin significantly induced the expression of the non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) in both p53 wild-type and p53-null cancer cell lines, suggesting that silibinin-induced NAG-1 up-regulation is p53-independent manner. Silibinin up-regulates early growth response-1 (EGR-1) expression. The ectopic expression of EGR-1 significantly increased NAG-1 promoter activity and NAG-1 protein expression in a dose-dependent manner. Furthermore, down-regulation of EGR-1 expression using siRNA markedly reduced silibinin-mediated NAG-1 expression, suggesting that the expression of EGR-1 is critical for silibinin-induced NAG-1 expression. We also observed that reactive oxygen species (ROS) are generated by silibinin; however, ROS did not affect silibinin-induced NAG-1 expression and apoptosis. In addition, we demonstrated that the mitogen-activated protein kinase (MAP kinase) signal transduction pathway is involved in silibinin-induced NAG-1 expression. Inhibitors of p38 MAP kinase (SB203580) attenuated silibinin-induced NAG-1 expression. Furthermore, we found that siRNA-mediated knockdown of NAG-1 attenuated silibinin-induced apoptosis. Collectively, the results of this study demonstrate for the first time that up-regulation of NAG-1 contributes to silibinin-induced apoptosis in cancer cells.