Kyung-Won Min

Damnacanthal-induced anti-inflammation is associated with inhibition of NF-κB activity.

Morinda citrifolia L. (Rubiaceae), commonly called noni, is a traditional folk medicinal plant with a long history of use for several diseases. Its anti-inflammation activity has been proposed, but detailed knowledge of this antiinflammation mechanism remains unclear. Here, we investigated the effects of noni extract and its major bioactive component damnacanthal on anti-inflammation in vivo as well as in vitro. Our data demonstrate that noni extract and its bioactive component damnacanthal exhibit suppression of inflammation as evidenced by the suppression of paw and ear edema in rats and mice, and down-regulation of lipopolysaccharide-induced nuclear factor-κB (NF-κB) activity, respectively. As a result, the expression of pro-cytokines, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were suppressed in the presence of damnacanthal. These results provide a potential use of damnacanthal in the treatment of inflammatory-related diseases.

NAG-1/GDF15 accumulates in the nucleus and modulates transcriptional regulation of the Smad pathway.

Protein dynamics, modifications and trafficking are all processes that can modulate protein activity. Accumulating evidence strongly suggests that many proteins have distinctive roles dependent on cellular location. Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) is a transforming growth factor-β (TGF-β) superfamily protein that has a role in cancer, obesity and inflammation. NAG-1 is synthesized and cleaved into a mature peptide, which is ultimately secreted into the extracellular matrix (ECM). In this study, we have found that full-length NAG-1 is expressed in not only the cytoplasm and ECM, but also in the nucleus. NAG-1 is dynamically moved to the nucleus, exported into cytoplasm and further transported into the ECM. We have also found that nuclear NAG-1 contributes to inhibition of the Smad pathway by interrupting the Smad complex. Overall, our study indicates that NAG-1 is localized in the nucleus and provides new evidence that NAG-1 controls transcriptional regulation in the Smad pathway.

A peroxisome proliferator-activated receptor ligand MCC-555 imparts anti-proliferative response in pancreatic cancer cells by PPARgamma-independent up-regulation of KLF4.

MCC-555 is a novel PPARα/γ dual ligand of the thiazolidinedione class and was recently developed as an anti-diabetic drug with unique properties. MCC-555 also has anti-proliferative activity through growth inhibition and apoptosis induction in several cancer cell types. Our group has shown that MCC-555 targets several proteins in colorectal tumorigenesis including nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1) which plays an important role in chemoprevention responsible for chemopreventive compounds. NAG-1 is a member of the TGF-β superfamily and is involved in tumor progression and development; however, NAG-1s roles in pancreatic cancer have not been studied. In this report, we found that MCC-555 alters not only NAG-1 expression, but also p21 and cyclin D1 expression. NAG-1 and p21 expression was not blocked by PPARγ-specific antagonist GW9662, suggesting that MCC-555-induced NAG-1 and p21 expression is independent of PPARγ activation. However, decreasing cyclin D1 by MCC-555 seems to be affected by PPARγ activation. Further, we found that the GC box located in the NAG-1 promoter play an important role in NAG-1 transactivation by MCC-555. Subsequently, we screened several transcription factors that may bind to the GC box region in the NAG-1 promoter and found that KLF4 potentially binds to this region. Expression of KLF4 precedes NAG-1 and p21 expression in the presence of MCC-555, whereas blocking KLF4 expression using specific KLF4 siRNA showed that both NAG-1 and p21 expression by MCC-555 was blocked. In conclusion, MCC-555s actions on anti-proliferation involve both PPARγ-dependent and -independent pathways, thereby enhancing anti-tumorigenesis in pancreatic cancer cells.

Moonlighting proteins in cancer

Since the 1980s, growing evidence suggested that the cellular localization of proteins determined their activity and biological functions. In a classical view, a protein is characterized by the single cellular compartment where it primarily resides and functions. It is now believed that when proteins appear in different subcellular locations, the cells surpass the expected activity of proteins given the same genomic information to fulfill complex biological behavior. Many proteins are recognized for having the potential to exist in multiple locations in cells. Dysregulation of translocation may cause cancer or contribute to poorer cancer prognosis. Thus, quantitative and comprehensive assessment of dynamic proteins and associated protein movements could be a promising indicator in determining cancer prognosis and efficiency of cancer treatment and therapy. This review will summarize these so-called moonlighting proteins, in terms of a coupled intracellular cancer signaling pathway. Determination of the detailed biological intracellular and extracellular transit and regulatory activity of moonlighting proteins permits a better understanding of cancer and identification of potential means of molecular intervention.

The involvement of endoplasmic reticulum stress in the suppression of colorectal tumorigenesis by tolfenamic acid.

The nonsteroidal anti-inflammatory drug tolfenamic acid has been shown to suppress cancer cell growth and tumorigenesis in different cancer models. However, the underlying mechanism by which tolfenamic acid exerts its antitumorigenic effect remains unclear. Previous data from our group and others indicate that tolfenamic acid alters expression of apoptosis- and cell-cycle arrest–related genes in colorectal cancer cells. Here, we show that tolfenamic acid markedly reduced the number of polyps and tumor load in APCmin/+ mice, accompanied with cyclin D1 downregulation in vitro and in vivo. Mechanistically, tolfenamic acid promotes endoplasmic reticulum (ER) stress, resulting in activation of the unfolded protein response (UPR) signaling pathway, of which PERK-mediated phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) induces the repression of cyclin D1 translation. Moreover, the PERK-eIF2α-ATF4 branch of the UPR pathway plays a role in tolfenamic acid-induced apoptosis in colorectal cancer cells, as silencing ATF4 attenuates tolfenamic acid-induced apoptosis. Taken together, these results suggest ER stress is involved in tolfenamic acid-induced inhibition of colorectal cancer cell growth, which could contribute to antitumorigenesis in a mouse model. Cancer Prev Res; 6(12); 1337–47. ©2013 AACR.

Hemostatic and Wound Healing Properties of Chromolaena odorata Leaf Extract.

Chromolaena odorata (L.) King and Robinson (Siam weed) extract has been used to stop bleeding and in wound healing in many tropical countries. However, its detailed mechanisms have not been elucidated. In this study, we examined the molecular mechanisms by which Siam weed extract (SWE) affected hemostatic and wound healing activities. SWE promoted Balb/c 3T3 fibroblast cell migration and proliferation. Subsequently, we found that heme oxygenase-1 (HO-1), the accelerating wound healing enzyme, was increased at the transcriptional and translational levels by SWE treatments. The HO-1 promoter analyzed with luciferase assay was also increased by treatment of SWE in a dose-dependent manner. This induction may be mediated by several kinase pathways including MEK, p38MAPK, AKT, and JNK. Quantitative real-time PCR using undifferentiated promonocytic cell lines revealed that thromboxane synthase (TXS), a potent vasoconstrictor and platelet aggregator, was increased and MMP-9, an anti platelet aggregator, was decreased in the presence of SWE. Our studies presented that SWE accelerated hemostatic and wound healing activities by altering the expression of genes, including HO-1, TXS, and MMP-9.

AUF1 facilitates microRNA-mediated gene silencing

Abstract Eukaryotic mRNA decay is tightly modulated by RNA-binding proteins (RBPs) and microRNAs (miRNAs). RBP AU-binding factor 1 (AUF1) has four isoforms resulting from alternative splicing and is critical for miRNA-mediated gene silencing with a distinct preference of target miRNAs. Previously, we have shown that AUF1 facilitates miRNA loading to Argonaute 2 (AGO2), the catalytic component of the RNA-induced silencing complex. Here, we further demonstrate that depletion of AUF1 abolishes the global interaction of miRNAs and AGO2. Single-molecule analysis revealed that AUF1 slowed down assembly of AGO2–let-7b–mRNA complex unexpectedly. However, target mRNAs recognized by both miRNA and AUF1 are less abundant upon AUF1 overexpression implying that AUF1 is a decay-promoting factor influencing multiple steps in AGO2–miRNA-mediated mRNA decay. Our findings indicate that AUF1 functions in promoting miRNA-mediated mRNA decay globally.

Disruption of the transforming growth factor-β pathway by tolfenamic acid via the ERK MAP kinase pathway

Transforming growth factor-β (TGF-β) modulates diverse cell physiological processes and plays a complicated role in tumor development. It has been well established that TGF-β inhibits cell proliferation in normal and early stage carcinoma and facilitates tumor metastasis in late-stage carcinoma. Therefore, blocking TGF-β signaling in advanced stage carcinogenesis provides a potentially interesting chemotherapeutic strategy. We aimed to determine the effect of tolfenamic acid (TA) on TGF-β-induced protumorigenic activity. Here, we demonstrate that TA attenuates tumor-promoting effects of TGF-β in cancer cells. Further observation indicates TA blocks the TGF-β/Smad pathway, and this blockage is mainly attributed to the interference of TGF-β1-driven phosphorylation of Smad2/3. We also show that TA could exert this effect on cancer cell lines from several different origins and that TA is much better than other non-steroidal anti-inflammatory drugs with respect to inhibition of TGF-β1-induced Smad2 phosphorylation. Finally, extracellular signal-regulated kinase mitogen-activated protein kinase plays a role in TA-induced suppression of Smad2/3 phosphorylation and subsequent nuclear accumulation of Smad2/3 in response to TGF-β1. Our study provides a possible mechanism by which TA affects anticancer activity by inhibiting the TGF-β pathway and sheds light on the application of TA for cancer patients.

A novel COX-independent mechanism of sulindac sulfide involves cleavage of epithelial cell adhesion molecule protein.

Non-steroidal anti-inflammatory drugs (NSAIDs) are extensively used over the counter to treat headaches and inflammation as well as clinically to prevent cancer among high-risk groups. The inhibition of cyclooxygenase (COX) activity by NSAIDs plays a role in their anti-tumorigenic properties. NSAIDs also have COX-independent activity which is not fully understood. In this study, we report a novel COX-independent mechanism of sulindac sulfide (SS), which facilitates a previously uncharacterized cleavage of epithelial cell adhesion molecule (EpCAM) protein. EpCAM is a type I transmembrane glycoprotein that has been implemented as an over-expressed oncogene in many cancers including colon, breast, pancreas, and prostate. We found EpCAM to be down-regulated by SS in a manner that is independent of COX activity, transcription regulation, de novo protein synthesis, and proteasomal degradation pathway. Our findings clearly demonstrate that SS drives cleavage of the extracellular portion of EpCAM near the N-terminus. This SS driven cleavage is blocked by a deleting amino acids 55-81 as well as simply mutating arginine residues at positions 80 and 81 to alanine of EpCAM. Proteolysis of EpCAM by SS may provide a novel mechanism by which NSAIDs affect anti-tumorigenesis at the post-translational level.

Nonsteroidal anti-inflammatory drug sulindac sulfide suppresses structural protein Nesprin-2 expression in colorectal cancer cells

BACKGROUND Nonsteroidal anti-inflammatory drugs (NSAIDs) are well known for treating inflammatory disease and have been reported to have anti-tumorigenic effects. Their mechanisms are not fully understood, but both cyclooxygenase (COX) dependent and independent pathways are involved. Our goal was to shed further light on COX-independent activity. METHODS Human colorectal cancer cells were observed under differential interference contrast microscopy (DICM), fluorescent microscopy, and micro-impedance measurement. Microarray analysis was performed using HCT-116 cells treated with sulindac sulfide (SS). PCR and Western blots were performed to confirm the microarray data and immunohistochemistry was performed to screen for Nesprin-2 expression. Micro-impedance was repeating including Nesprin-2 knock-down by siRNA. RESULTS HCT-116 cells treated with SS showed dramatic morphological changes under DICM and fluorescent microscopy, as well as weakened cellular adhesion as measured by micro-impedance. Nesprin-2 was selected from two independent microarrays, based on its novelty in relation to cancer and its role in cell organization. SS diminished Nesprin-2 mRNA expression as assessed by reverse transcriptase and real time PCR. Various other NSAIDs were also tested and demonstrated that inhibition of Nesprin-2 mRNA was not unique to SS. Additionally, immunohistochemistry showed higher levels of Nesprin-2 in many tumors in comparison with normal tissues. Further micro-impedance experiments on cells with reduced Nesprin-2 expression showed a proportional loss of cellular adhesion. CONCLUSIONS Nesprin-2 is down-regulated by NSAIDs and highly expressed in many cancers. GENERAL SIGNIFICANCE Our data suggest that Nesprin-2 may be a potential novel oncogene in human cancer cells and NSAIDs could decrease its expression.