Nup210 promotes colorectal cancer progression by regulating nuclear plasma transport.

The nuclear pore complex (NPC) regulates nucleoplasmic transport, transcription and genomic integrity in eukaryotic cells. However, little is known about how NPC works in cancer. In this study, we investigated the role of the nuclear pore protein 210 (Nucleoporin 210, Nup210) in colorectal cancer (CRC). Bioinformatics analysis revealed that the expression of Nup210 was increased in CRC and was associated with poor patient prognosis, but it was not a statistically significant independent prognostic factor. Moreover, knockdown of Nup210 in CRC cells inhibited the proliferation, invasion and metastasis of CRC cells in vivo and in vitro. Additionally, nuclear size and nuclear plasma material transport capacity decreased along with the number and density of NPCs on the surface of CRC cells when Nup210 expression was inhibited. Furthermore, Nup210 required nuclear localization sequences (NLS) to localize to the nuclear membrane surface and interact with importin α/ß, which in turn affected the transit of nuclear plasma material. Importazole, a small molecule inhibitor of importin, along with therapy that targets the Nup210 protein is anticipated to be a novel strategy for CRC treatment. Their combination may be able to more effectively lower CRC tumor load. In conclusion, Nup210 modulates cellular nucleoplasmic transport capability and cell surface NPC density via NLS, thus promoting CRC progression. This discovery validates the molecular function of NPC in the development of CRC and provides a theoretical foundation for NPC-regulated nuclear import targeting as a therapeutic strategy for CRC.

Golgin45 assists mitosis via its nuclear localization sequence.

In mammalian cells, the Golgi apparatus undergoes fragmentation for its correct partition into two daughter cells during mitosis. Several Golgi structural proteins have been demonstrated to regulate Golgi disassembly/reassembly and spindle formation. However, it is largely unknown whether Golgi proteins mediate other major events in mitosis. Here, we report that Golgin45, a Golgi tethering protein, participates in recruiting PLK1 to the kinetochores. Upon entry into mitosis, Golgin45 binds PLK1 and a nuclear import protein, importin ß2. Enriched RanGTP at kinetochores in prometaphase and metaphase sequesters importin ß2 from Golgin45 and liberates Golgin45-PLK1 complex, which then gets further delivered to the kinetochores by Golgin45-KNL1 interaction. R375A mutation in Golgin45 that specifically disrupts Golgin45-importin ß2 interaction impairs PLK1 localization to the kinetochores, leading to mitotic arrest. Our findings reveal a novel role of a golgin tether protein in mediating Ran-dependent PLK1 enrichment on the kinetochores for proper progression of mitosis.

Thermal Stress and Nuclear Transport.

Nuclear transport is the basis for the biological reaction of eukaryotic cells, as it is essential to coordinate nuclear and cytoplasmic events separated by nuclear envelope. Although we currently understand the basic molecular mechanisms of nuclear transport in detail, many unexplored areas remain. For example, it is believed that the regulations and biological functions of the nuclear transport receptors (NTRs) highlights the significance of the transport pathways in physiological contexts. However, physiological significance of multiple parallel transport pathways consisting of more than 20 NTRs is still poorly understood, because our knowledge of each pathway, regarding their substrate information or how they are differently regulated, is still limited. In this report, we describe studies showing how nuclear transport systems in general are affected by temperature rises, namely, thermal stress or heat stress. We will then focus on Importin α family members and unique transport factor Hikeshi, because these two NTRs are affected in heat stress. Our present review will provide an additional view to point out the importance of diversity of the nuclear transport pathways in eukaryotic cells.

Single Acetylation-mimetic Mutation in TDP-43 Nuclear Localization Signal Disrupts Importin α1/ß Signaling.

Cytoplasmic aggregation of the TAR-DNA binding protein of 43 kDa (TDP-43) is the hallmark of sporadic amyotrophic lateral sclerosis (ALS). Most ALS patients with TDP-43 aggregates in neurons and glia do not have mutations in the TDP-43 gene but contain aberrantly post-translationally modified TDP-43. Here, we found that a single acetylation-mimetic mutation (K82Q) near the TDP-43 minor Nuclear Localization Signal (NLS) box, which mimics a post-translational modification identified in an ALS patient, can lead to TDP-43 mislocalization to the cytoplasm and irreversible aggregation. We demonstrate that the acetylation mimetic disrupts binding to importins, halting nuclear import and preventing importin α1/ß anti-aggregation activity. We propose that perturbations near the NLS are an additional mechanism by which a cellular insult other than a genetically inherited mutation leads to TDP-43 aggregation and loss of function. Our findings are relevant to deciphering the molecular etiology of sporadic ALS.

Nuclear localization of Arabidopsis HD-Zip IV transcription factor GLABRA2 is driven by importin α.

GLABRA2 (GL2), a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor from Arabidopsis, is a developmental regulator of specialized cell types in the epidermis. GL2 contains a monopartite nuclear localization sequence (NLS) that is conserved in most HD-Zip IV members across the plants. We demonstrate that NLS mutations affect nuclear transport and result in a loss-of-function phenotypes. NLS fusions to enhanced yellow fluorescent protein (EYFP) show that it is sufficient for nuclear localization in roots and trichomes. Despite partial overlap of the NLS with the homeodomain, genetic dissection indicates that nuclear localization and DNA binding are separable functions. Affinity purification of GL2 from plants followed by MS-based proteomics identified importin α (IMPα) isoforms as potential GL2 interactors. NLS structural prediction and molecular docking studies with IMPα-3 revealed major interacting residues. Cytosolic yeast two-hybrid assays and co-immunoprecipitation experiments with recombinant proteins verified NLS-dependent interactions between GL2 and several IMPα isoforms. IMPα triple mutants (impα-1,2,3) exhibit abnormal trichome formation and defects in GL2 nuclear localization in trichomes, consistent with tissue-specific and redundant functions of IMPα isoforms. Taken together, our findings provide mechanistic evidence for IMPα-dependent nuclear localization of GL2 in Arabidopsis, a process that is critical for cell type differentiation of the epidermis.

Nucleoporin Nup98 is an essential factor for ipo4 dependent protein import.

Nucleocytoplasmic transport of macromolecules is essential in eukaryotic cells. In this process, the karyopherins play a central role when they transport cargoes across the nuclear pore complex. Importin 4 belongs to the karyopherin ß family. Many studies have focused on finding substrates for importin 4, but no direct mechanism studies of its precise transport function have been reported. Therefore, this paper mainly aimed to study the mechanism of nucleoporins in mediating nuclear import and export of importin 4. To address this question, we constructed shRNAs targeting Nup358, Nup153, Nup98, and Nup50. We found that depletion of Nup98 resulted in a shift in the subcellular localization of importin 4 from the cytoplasm to the nucleus. Mutational analysis demonstrated that Nup98 physically and functionally interacts with importin 4 through its N-terminal phenylalanine-glycine (FG) repeat region. Mutation of nine of these FG motifs to SG motifs significantly attenuated the binding of Nup98 to importin 4, and we further confirmed the essential role of the six FG motifs in amino acids 121-360 of Nup98 in binding with importin 4. In vitro transport assay also confirmed that VDR, the substrate of importin 4, could not be transported into the nucleus after Nup98 knockdown. Overall, our results showed that Nup98 is required for efficient importin 4-mediated transport. This is the first study to reveal the mechanism of importin 4 in transporting substrates into the nucleus.

Serotype-Specific Regulation of Dengue Virus NS5 Protein Subcellular Localization.

Dengue virus (DENV) nonstructural protein 5 (NS5), consisting of methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, is critical for viral RNA synthesis within endoplasmic reticulum-derived replication complexes in the cytoplasm. However, a significant proportion of NS5 is localized to the nucleus of infected cells for DENV2, 3, and 4, whereas DENV1 NS5 is localized diffusely in the cytoplasm. We still have an incomplete understanding of how the DENV NS5 subcellular localization is regulated. Within NS5, two putative nuclear localization signal (NLS) sequences have been identified: NLSCentral residing in the palm of the RdRp domain as well as the recently discovered NLSC-term residing in the flexible region at the C-terminal of the RdRp domain. We have previously shown that DENV2 NS5 nuclear localization can be significantly reduced by single-point mutations to the NLSC-term. Here, we present biochemical, virological, and structural data demonstrating that the relative importance of either NLS in NS5 nuclear localization is unique to each of the four DENV serotypes. DENV1 NS5's cytoplasmic localization appears to be due to a functionally weak interaction between its NLSCentral and importin-α (IMPα), while DENV2 NS5 is almost exclusively nuclear through its NLSC-term's strong interaction with IMPα. Both NLSs of DENV3 NS5 appear to contribute to directing its nuclear localization. Lastly, in the case of DENV4, the regulation of its NS5 nuclear localization remains an enigma but appears to be associated with its NLSC-term.

A novel disulfide death-related genes prognostic signature identifies the role of IPO4 in glioma progression.

BACKGROUND: "Disulfide death," a form of cellular demise, is triggered by the abnormal accumulation of intracellular disulfides under conditions of glucose deprivation. However, its role in the prognosis of glioma remains undetermined. Therefore, the main objective of this study is to establish prognostic signature based on disulfide death-related genes (DDRGs) and to provide new solutions in choosing the effective treatment of glioma. METHODS: The RNA transcriptome, clinical information, and mutation data of glioma samples were sourced from The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA), while normal samples were obtained from the Genotype-Tissue Expression (GTEx). DDRGs were compiled from previous studies and selected through differential analysis and univariate Cox regression analysis. The molecular subtypes were determined through consensus clustering analysis. Further, LASSO analysis was employed to select characteristic genes, and subsequently, a risk model comprising seven DDRGs was constructed based on multivariable Cox analysis. Kaplan-Meier survival curves were employed to assess survival differences between high and low-risk groups. Additionally, functional analyses (GO, KEGG, GSEA) were conducted to explore the potential biological functions and signaling pathways of genes associated with the model. The study also explored immune checkpoint (ICP) genes, immune cell infiltration levels, and immune stromal scores. Finally, the effect of Importin-4(IPO4) on glioma has been further confirmed through RT-qPCR, Western blot, and cell functional experiments. RESULTS: 7 genes associated with disulfide death were obtained and two subgroups of patients with different prognosis and clinical characteristics were identified. Risk signature was subsequently developed and proved to serve as an prognostic predictor. Notably, the high-risk group exhibited an immunosuppressive microenvironment characterized by a high concentration of M2 macrophages and regulatory T cells (Tregs). In contrast, the low-risk group showed lower half-maximal inhibitory concentration (IC50) values. Therefore, patients in the high-risk group may benefit more from immunotherapy, while patients in the low-risk group may benefit more from chemotherapy. In addition, in vitro experiments have shown that inhibition of the expression of IPO4 leads to a significant reduction in the proliferation, migration, and invasion of glioma cells. CONCLUSION: This study identified two glioma subtypes and constructed a prognostic signature based on DDRGs. The signature has the potential to optimize the selection of patients for immune- and chemotherapy and provided a potential therapeutic target for glioma.

SAD2 functions in plant pathogen Pseudomonas syringae pv tomato DC3000 defense by regulating the nuclear accumulation of MYB30 in Arabidopsis thaliana.

Accurate nucleocytoplasmic transport of signal molecules is essential for plant growth and development. Multiple studies have confirmed that nucleocytoplasmic transport and receptors are involved in regulating plant disease resistance responses, however, little is known about the regulatory mechanism in plants. In this study, we showed that the mutant of the importin beta-like protein SAD2 exhibited a more susceptible phenotype than wild-type Col-0 after treatment with Pseudomonas syringae pv tomato DC3000 (Pst DC3000). Coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) experiments demonstrated that SAD2 interacts with the hypersensitive response (HR)-positive transcriptional regulator MYB30. Subcellular localization showed that MYB30 was not fully localized in the nucleus in sad2-5 mutants, and western-blot experiments further indicated that SAD2 was required for MYB30 nuclear trafficking during the pathogen infection process. A phenotypic test of pathogen inoculation demonstrated that MYB30 partially rescued the disease symptoms of sad2-5 caused by Pst DC3000, and that MYB30 worked downstream of SAD2 in plant pathogen defense. These results suggested that SAD2 might be involved in plant pathogen defense by mediating MYB30 nuclear trafficking. Taken together, our results revealed the important function of SAD2 in plant pathogen defense and enriched understanding of the mechanism of nucleocytoplasmic transport-mediated plant pathogen defense.

Coronatine orchestrates ABI1-mediated stomatal opening to facilitate bacterial pathogen infection through importin ß protein SAD2.

Stomatal immunity plays an important role during bacterial pathogen invasion. Abscisic acid (ABA) induces plants to close their stomata and halt pathogen invasion, but many bacterial pathogens secrete phytotoxin coronatine (COR) to antagonize ABA signaling and reopen the stomata to promote infection at early stage of invasion. However, the underlining mechanism is not clear. SAD2 is an importin ß family protein, and the sad2 mutant shows hypersensitivity to ABA. We discovered ABI1, which negatively regulated ABA signaling and reduced plant sensitivity to ABA, was accumulated in the plant nucleus after COR treatment. This event required SAD2 to import ABI1 to the plant nucleus. Abolition of SAD2 undermined ABI1 accumulation. Our study answers the long-standing question of how bacterial COR antagonizes ABA signaling and reopens plant stomata during pathogen invasion.

Karyopherin α2 is a maternal effect gene required for early embryonic development and female fertility in mice.

The nuclear transport of proteins plays an important role in mediating the transition from egg to embryo and distinct karyopherins have been implicated in this process. Here, we studied the impact of KPNA2 deficiency on preimplantation embryo development in mice. Loss of KPNA2 results in complete arrest at the 2cell stage and embryos exhibit the inability to activate their embryonic genome as well as a severely disturbed nuclear translocation of Nucleoplasmin 2. Our findings define KPNA2 as a new maternal effect gene.

Ivermectin ameliorates acute myocarditis via the inhibition of importin-mediated nuclear translocation of NF-κB/p65.

BACKGROUND: Myocarditis is an important clinical issue which lacks specific treatment by now. Ivermectin (IVM) is an inhibitor of importin α/ß-mediated nuclear translocation. This study aimed to explore the therapeutic effects of IVM on acute myocarditis. METHODS: Mouse models of coxsackie B3 virus (CVB3) infection-induced myocarditis and experimental autoimmune myocarditis (EAM) were established to evaluate the effects of IVM. Cardiac functions were evaluated by echocardiography and Millar catheter. Cardiac inflammatory infiltration was assessed by histological staining. Cytometric bead array and quantitative real-time PCR were used to detect the levels of pro-inflammatory cytokines. The macrophages and their M1/M2 polarization were analyzed via flow cytometry. Protein expression and binding were detected by co-immunoprecipitation, Western blotting and histological staining. The underlying mechanism was verified in vitro using CVB3-infected RAW264.7 macrophages. Cyclic polypeptide (cTN50) was synthesized to selectively inhibit the nuclear translocation of NF-κB/p65, and CVB3-infected RAW264.7 cells were treated with cTN50. RESULTS: Increased expression of importin ß was observed in both models. IVM treatment improved cardiac functions and reduced the cardiac inflammation associated with CVB3-myocarditis and EAM. Furthermore, the pro-inflammatory cytokine (IL-1ß/IL-6/TNF-α) levels were downregulated via the inhibition of the nuclear translocation of NF-κB/p65 in macrophages. IVM and cTN50 treatment also inhibited the nuclear translocation of NF-κB/p65 and downregulated the expression of pro-inflammatory cytokines in RAW264.7 macrophages. CONCLUSIONS: Ivermectin inhibits the nuclear translocation of NF-κB/p65 and the expression of major pro-inflammatory cytokines in myocarditis. The therapeutic effects of IVM on viral and non-viral myocarditis models suggest its potential application in the treatment of acute myocarditis.

Apoptosis-inducing factor 1 mediates Vibrio splendidus-induced coelomocyte apoptosis via importin ß dependent nuclear translocation in Apostichopus japonicus.

As is well known, apoptosis is an important form of immune response and immune regulation, particularly playing a crucial role in combating microbial infections. Apoptosis-inducing factor 1 (AIF-1) is essential for apoptosis to induce chromatin condensation and DNA fragmentation via a caspase-independent pathway. The nuclear translocation of AIF-1 is a key step in apoptosis but the molecular mechanism is still unclear. In this study, the homologous gene of AIF-1, named AjAIF-1, was cloned and identified in Apostichopus japonicus. The mRNA expression of AjAIF-1 was significantly increased by 46.63-fold after Vibrio splendidus challenge. Silencing of AjAIF-1 was found to significantly inhibit coelomocyte apoptosis because the apoptosis rate of coelomocyte decreased by 0.62-fold lower compared with the control group. AjAIF-1 was able to promote coelomocyte apoptosis through nuclear translocation under the V. splendidus challenge. Moreover, AjAIF-1 and Ajimportin ß were mainly co-localized around the nucleus in vivo and silencing Ajimportin ß significantly inhibited the nuclear translocation of AjAIF-1 and suppressed coelomocyte apoptosis by 0.64-fold compared with control. In summary, nuclear translocation of AjAIF-1 will likely mediate coelomocyte apoptosis through an importin ß-dependent pathway in sea cucumber.

A bipartite NLS motif mediates the nuclear import of Drosophila moesin.

The ERM protein family, which consists of three closely related proteins in vertebrates, ezrin, radixin, and moesin (ERM), is an ancient and important group of cytoplasmic actin-binding and organizing proteins. With their FERM domain, ERMs bind various transmembrane proteins and anchor them to the actin cortex through their C-terminal F-actin binding domain, thus they are major regulators of actin dynamics in the cell. ERMs participate in many fundamental cellular processes, such as phagocytosis, microvilli formation, T-cell activation and tumor metastasis. We have previously shown that, besides its cytoplasmic activities, the single ERM protein of Drosophila melanogaster, moesin, is also present in the cell nucleus, where it participates in gene expression and mRNA export. Here we study the mechanism by which moesin enters the nucleus. We show that the nuclear import of moesin is an NLS-mediated, active process. The nuclear localization sequence of the moesin protein is an evolutionarily highly conserved, conventional bipartite motif located on the surface of the FERM domain. Our experiments also reveal that the nuclear import of moesin does not require PIP2 binding or protein activation, and occurs in monomeric form. We propose, that the balance between the phosphorylated and non-phosphorylated protein pools determines the degree of nuclear import of moesin.

Non-Canonical Localization of Cardiac Troponins: Expanding Functions or Causing Pathologies?

The troponin complex-consisting of three subunits: troponin C (TnC), cardiac troponin I (cTnI) and cardiac troponin T (cTnT)-plays a key role in the regulation of myocardial contraction. Troponins are preferentially localized in the cytoplasm and bind to myofibrils. However, numerous, albeit scattered, studies have shown the presence of troponins in the nuclei of muscle cells. There is increasing evidence that the nuclear localization of troponins may be functionally important, making troponins an important nuclear player in the pathogenesis of various diseases including cancer and myopathies. Further studies in this area could potentially lead to the development of treatments for certain pathologies. In this review, we collected and discussed recent data on the properties of non-canonically localized cardiac troponins, the molecular mechanisms leading to this non-canonical localization, and the possible functions or pathological effects of these non-canonically localized troponins.

Drosophila Importin Alpha 1 (Dα1) Is Required to Maintain Germline Stem Cells in the Testis Niche.

Stem cell maintenance and differentiation can be regulated via the differential activity of transcription factors within stem cells and their progeny. For these factors to be active, they need to be transported from their site of synthesis in the cytoplasm into the nucleus. A tissue-specific requirement for factors involved in nuclear importation is a potential mechanism to regulate stem cell differentiation. We have undertaken a characterization of male sterile importin alpha 1 (Dα1) null alleles in Drosophila and found that Dα1 is required for maintaining germline stem cells (GSCs) in the testis niche. The loss of GSCs can be rescued by ectopic expression of Dα1 within the germline but the animals are still infertile, indicating a second role for Dα1 in spermatogenesis. Expression of a Dα1 dominant negative transgene in GSCs confirmed a functional requirement for Dα1 in GSC maintenance but expression of the transgene in differentiating spermatogonia did not exhibit a phenotype indicating a specific role for Dα1 within GSCs. Our data indicate that Dα1 is utilized as a regulatory protein within GSCs to facilitate nuclear importation of proteins that maintain the stem cell pool.

Structural basis for nuclear import of bat adeno-associated virus capsid protein.

Adeno-associated viruses (AAV) are one of the world's most promising gene therapy vectors and as a result, are one of the most intensively studied viral vectors. Despite a wealth of research into these vectors, the precise characterisation of AAVs to translocate into the host cell nucleus remains unclear. Recently we identified the nuclear localization signals of an AAV porcine strain and determined its mechanism of binding to host importin proteins. To expand our understanding of diverse AAV import mechanisms we sought to determine the mechanism in which the Cap protein from a bat-infecting AAV can interact with transport receptor importins for translocation into the nucleus. Using a high-resolution crystal structure and quantitative assays, we were able to not only determine the exact region and residues of the N-terminal domain of the Cap protein which constitute the functional NLS for binding with the importin alpha two protein, but also reveal the differences in binding affinity across the importin-alpha isoforms. Collectively our results allow for a detailed molecular view of the way AAV Cap proteins interact with host proteins for localization into the cell nucleus.

Nuclear-import receptors as gatekeepers of pathological phase transitions in ALS/FTD.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders on a disease spectrum that are characterized by the cytoplasmic mislocalization and aberrant phase transitions of prion-like RNA-binding proteins (RBPs). The common accumulation of TAR DNA-binding protein-43 (TDP-43), fused in sarcoma (FUS), and other nuclear RBPs in detergent-insoluble aggregates in the cytoplasm of degenerating neurons in ALS/FTD is connected to nuclear pore dysfunction and other defects in the nucleocytoplasmic transport machinery. Recent advances suggest that beyond their canonical role in the nuclear import of protein cargoes, nuclear-import receptors (NIRs) can prevent and reverse aberrant phase transitions of TDP-43, FUS, and related prion-like RBPs and restore their nuclear localization and function. Here, we showcase the NIR family and how they recognize cargo, drive nuclear import, and chaperone prion-like RBPs linked to ALS/FTD. We also discuss the promise of enhancing NIR levels and developing potentiated NIR variants as therapeutic strategies for ALS/FTD and related neurodegenerative proteinopathies.

Importin α2 participates in RNA interference against bamboo mosaic virus accumulation in Nicotiana benthamiana via NbAGO10a-mediated small RNA clearance.

Karyopherins, the nucleocytoplasmic transporters, participate in multiple RNA silencing stages by transporting associated proteins into the nucleus. Importin α is a member of karyopherins and has been reported to facilitate virus infection via nuclear import of viral proteins. Unlike other RNA viruses, silencing of importin α2 (α2i) by virus-induced gene silencing (VIGS) boosted the titre of bamboo mosaic virus (BaMV) in protoplasts, and inoculated and systemic leaves of Nicotiana benthamiana. The enhanced BaMV accumulation in importin α2i plants was linked to reduced levels of RDR6-dependent secondary virus-derived small-interfering RNAs (vsiRNAs). Small RNA-seq revealed importin α2 silencing did not affect the abundance of siRNAs derived from host mRNAs but significantly reduced the 21 and 22 nucleotide vsiRNAs in BaMV-infected plants. Deletion of BaMV TGBp1, an RNA silencing suppressor, compromised importin α2i-mediated BaMV enhancement. Moreover, silencing of importin α2 upregulated NbAGO10a, a proviral protein recruited by TGBp1 for BaMV vsiRNAs clearance, but hindered the nuclear import of NbAGO10a. Taken together, these results indicate that importin α2 acts as a negative regulator of BaMV invasion by controlling the expression and nucleocytoplasmic shuttling of NbAGO10a, which removes vsiRNAs via the TGBp1-NbAGO10a-SDN1 pathway. Our findings reveal the hidden antiviral mechanism of importin α2 in countering BaMV infection in N. benthamiana.

Linderapyrone analogue LPD-01 as a cancer treatment agent by targeting importin7.

The Wnt/ß-catenin signaling pathway plays important roles in several cancer cells, including cell proliferation and development. We previously succeeded in synthesizing a small molecule compound inhibiting the Wnt/ß-catenin signaling pathway, named LPD-01 (1), and 1 inhibited the growth of human colorectal cancer (HT-29) cells. In this study, we revealed that 1 inhibits the growth of HT-29 cells stronger than that of another human colorectal cancer (SW480) cells. Therefore, we have attempted to identify the target proteins of 1 in HT-29 cells. Firstly, we investigated the effect on the expression levels of the Wnt/ß-catenin signaling pathway-related proteins. As a result, 1 inhibited the expression of target proteins of Wnt/ß-catenin signaling pathway (c-Myc and Survivin) and their genes, whereas the amount of transcriptional co-activator (ß-catenin) was not decreased, suggesting that 1 inhibited the Wnt/ß-catenin signaling pathway without affecting ß-catenin. Next, we investigated the target proteins of 1 using magnetic FG beads. Chemical pull-down assay combined with mass spectrometry suggested that 1 directly binds to importin7. As expected, 1 inhibited the nuclear translocation of importin7 cargoes such as Smad2 and Smad3 in TGF-ß-stimulated HT-29 cells. In addition, the knockdown of importin7 by siRNA reduced the expression of target genes of Wnt/ß-catenin signaling pathway. These results suggest that importin7 is one of the target proteins of 1 for inhibition of the Wnt/ß-catenin signaling pathway.