Discovery of a highly potent and orally available importin-β1 inhibitor that overcomes enzalutamide-resistance in advanced prostate cancer

Nuclear transporter importin-β1 is emerging as an attractive target by virtue of its prevalence in many cancers. However, the lack of druggable inhibitors restricts its therapeutic proof of concept. In the present work, we optimized a natural importin-β1 inhibitor DD1 to afford an improved analog DD1-Br with better tolerability (>25 folds) and oral bioavailability. DD1-Br inhibited the survival of castration-resistant prostate cancer (CRPC) cells with sub-nanomolar potency and completely prevented tumor growth in resistant CRPC models both in monotherapy (0.5 mg/kg) and in enzalutamide-combination therapy. Mechanistic study revealed that by targeting importin-β1, DD1-Br markedly inhibited the nuclear accumulation of multiple CRPC drivers, particularly AR-V7, a main contributor to enzalutamide resistance, leading to the integral suppression of downstream oncogenic signaling. This study provides a promising lead for CRPC and demonstrates the potential of overcoming drug resistance in advanced CRPC via targeting importin-β1.

The nucleocapsid protein of rice stripe virus in cell nuclei of vector insect regulates viral replication

Rice stripe virus (RSV) transmitted by the small brown planthopper causes severe rice yield losses in Asian countries. Although viral nuclear entry promotes viral replication in host cells, whether this phenomenon occurs in vector cells remains unknown. Therefore, in this study, we systematically evaluated the presence and roles of RSV in the nuclei of vector insect cells. We observed that the nucleocapsid protein (NP) and viral genomic RNAs were partially transported into vector cell nuclei by utilizing the importin α nuclear transport system. When blocking NP nuclear localization, cytoplasmic RSV accumulation significantly increased. In the vector cell nuclei, NP bound the transcription factor YY1 and affected its positive regulation to FAIM. Subsequently, decreased FAIM expression triggered an antiviral caspase-dependent apoptotic reaction. Our results reveal that viral nuclear entry induces completely different immune effects in vector and host cells, providing new insights into the balance between viral load and the immunity pressure in vector insects.

Role of HIF1α Regulatory Factors in Stem Cells

Hypoxia-inducible factor 1 (HIF1) is a master transcription factor that induces the transcription of genes involved in the metabolism and behavior of stem cells. HIF1-mediated adaptation to hypoxia is required to maintain the pluripotency and survival of stem cells under hypoxic conditions. HIF1 activity is well known to be tightly controlled by the alpha subunit of HIF1 (HIF1α). Understanding the regulatory mechanisms that control HIF1 activity in stem cells will provide novel insights into stem cell biology under hypoxia. Recent research has unraveled the mechanistic details of HIF1α regulating processes, suggesting new strategies for regulating stem cells. This review summarizes recent experimental studies on the role of several regulatory factors (including calcium, 2-oxoglutarate-dependent dioxygenase, microtubule network, importin, and coactivators) in regulating HIF1α activity in stem cells.

Effect of Signal-transducing Adaptor Protein 2 and Importin 5 Expression on Skeletal Muscle Protein Turnover / 中国康复理论与实践

@# Objective To explore the expression of signal-transducing adaptor protein 2 (STAP2) and importin 5 (Ipo5), and the effect of them on skeletal muscle protein turnover. Methods Twelve male Sprague-Dawley rats were divided into control group (n=6) and model group (n=6). The model group established skeletal muscle hypertrophy induced by blood flow restriction. The soleus muscle cells in both groups were cultured in vitro, and those in the model group infected with shRNA lentivirus to make the expression of STAP2 and Ipo5 silencing, over-expression and gene function salvage finally. The expression of STAP2, Ipo5 and myostatin were determined with Western blotting in soleus muscle in both groups.Results The expression of STAP2 increased (t=-11.786, P<0.001), while the expression of Ipo5 and myostatin decreased (t>14.039, P<0.001) in the model groups compared with those in the control group. Under the control group and the model group, and target genes silencing group, overexpressing group and rescuing group, the expression of myostatin increased with STAP2 decrease and Ipo5 increase.Conclusion The expression of STAP2 increasing and Ipo5 decreasing would promote the protein synthesis.

Effects of rs35100176 polymorphism in IPO8 promoter on gene expression / 中国病理生理杂志

AIM:To investigate the effect of rs 35100176 CCT insertion/deletion polymorphism in the promoter region of importin 8 ( IPO8) gene on its mRNA expression .METHODS:A 342-bp fragment of IPO8 gene promoter con-taining the rs35100176 polymorphism was amplified from 49 DNA samples and sequenced .The IPO8 promoter fragments containing CCT 3-nucleotide insertion or deletion were amplified using the corresponding homozygote DNA samples .The PCR products were sequenced and inserted into the luciferase reporter vector pGL 3-Basic.Recombinant vectors were trans-fected into the cells by Fugene 6.0 and the expression of the reporter gene was detected by a dual-luciferase reporter assay system.The mRNA expression level of IPO8 was detected by real-time PCR in 3-nucleotide insertion or deletion homozygote cells.RESULTS:The sequencing results showed that there were 3 kinds of genotypes in the rs35100176 polymorphism, CCT/CCT, CCT/-and -/-, and the gene frequencies were 18.37%, 55.10% and 26.53%, respectively.The re-combinant expression vectors pGL 3-3N Insertion and pGL3-3N Deletion were successfully constructed .The luciferase assay showed that pGL3-3N Insertion produced significantly lower luciferase activity than that by pGL 3-3N Deletion.Real-time PCR showed that HEK293 cells with 3-nucleotide insertion homozygote expressed relative lower IPO 8 mRNA than Saos-2 cells with 3-nucleotide deletion homozygote .CONCLUSION: The CCT 3-nucleotide insertion variant decreases the pro-moter activity of IPO8, thus affecting the gene expression .

Differential expression of nucleus-cytoplasm transport receptor protein importin13 in limbal neoplasms / 中华实验眼科杂志

Background The study on normal stem cell markers provides a new way of thinking of that pathogenesis of cancer research and looking for specific markers of cancer stem cells.Importin13 (IPO13) is a novel nucleus-cytoplasm transport receptor protein of importin β family,the study on the biological behavior of IPO13 in ocular tissue and limbal neoplasms is lacking.Objective This study was to investigate the differential expression of IPO13 and p63 in human benign and malignant conjunctiva-cornea neoplasms.Methods The specimens of normal donor limbal and conjunctival tissues (6),conjunciva-cornea papilloma (CCP) (6) and conjunctiva-cornea intraepithelial neoplasia(CCIN) (6)were collected from Xiangya Hospital of Center South University.The expressions of IPO13 and p63 in the corresponding tissue were qualitatively and quantitatively detected using immunochemistry.The A values of IPO13 and p63 positive response were calculated and compared among the 3 types specimens.Results The immunohistostaining on frozen sections showed that IPO13 was expressed in nuclei of basal cells of limbal and conjunctival epithelium and the cellular nuclei of basal cells and suprabasal cells of CCP and CCIN epithelium.The A values of IPO13 positive expression were 1687± 1014,3546± 1375 and 7635 ±2854 in the normal limbal specimen,CCP and CCIN,respectively,showing a significant difference among them (F =7.23,P＜0.05),and the A value was higher in the CCP and CCIN than that in the normal limbal tissue (q =4.02,5.13,P＜0.05),and that in the CCIN was significantly elevated in comparison with CCP(q =3.45,P＜0.05).p63 was expressed in nuclei of basal cells and suprabasal of limbal,conjunctival and CCP epithelium,and was expressed in nuclei of the entire CCIN epithelium.The expressions of p63 in the normal conjunctiva-cornea tissue,CCP and CCIN were 2110± 1229,3966± 2129 and 6650± 2136 respectively,with significant difference among the three different specimens (F =6.17,P＜ 0.05),and the A value of p63 positive expression was significantly elevated in the CCP and CCIN compared with normal limbal specimen (q =4.33,5.01,P＜0.05),and that in the CCIN was significantly elevated in comparison with CCP(q=3.83,P＜0.05).Conclusions IPO13 is more specifical in marking the poorly differentiated cells in limbal epithelial proliferative lesions than p63.Compared with the normal limbal specimen,the expressions of IPO13 and p63 in CCP and CCIN specimens gradually upregulat,which suggests that IPO13 and p63 may play a positive regulatory role in conjunctiva-cornea proliferative lesions.The differential expression of IPO13 and p63 is predominant between benign and malignant limbal epithelial proliferative lesions,indicating that IPO13 and p63 may play an important role in regulating the abnormal proliferation and differentiation of limbal epithelial proliferative lesions.

Polypeptide cSN50 inhibits the expression of NF-κB and its downstream factor via Importin α in HepG2 cells / 中国综合临床

Objective To investigate whether polypeptide cSN50,as a transmembrane peptides,can inhibit NF-κB nuclear translocation and its downstream gene expression to play a role to protect cells by blocking the combination of NF-κB with the importinα3 during the alcohol and endotoxin-induced inflammation and apoptosis.Methods Flow cytometry method was used to observe the apoptosis rate of HepG2 by different concentration of alcohol and/or endotoxin.Spectrophotometer method was used to detect Caspase-3 activity.TNF- α in cell culture supernatant was detected by ELISA assay.p50,importinα3,and IκBαunder the stimulation of alcohol and/or endotoxin at selected optimal concentration were detected by Western blot.Indirect immunofluorescence assay was used to measure the activation of p50,IκBα,and importinα3.Results The change of TNF-α and Caspase-3 in the dose-effect course,compared with control group,was significant (P ＜0.05 ).p50 was increased in the nucleus ( P ＜ 0.05 ),and IκBα was decreased in the cytoplasm ( P ＜ 0.05 ).cSN50( 100 μmol/L) partially blocked nuclear translocation of p50 and its downstream factor( P ＜ 0.05 ).Conclusion NF-κB nuclear translocation may play an important role in acute alcoholic liver injury.cSN50 can effectively inhibit NF-κB activity and its downstream gene expression in HepG2 cells.

Deletion of the Importin-alpha Gene in the Breast Cancer Cell

BACKGROUND: BRCA1 (breast-cancer gene 1) is a tumor suppressor gene that accounts for nearly all families of both early onset breast and ovarian cancer and about 45% of families with breast cancer only. Sporadic nonhereditary breast cancer is recognized as the most common form of this malignancy. However, presence of germ-line mutations in the BRCA1 gene of these tumors is an infrequent event. The BRCA1 protein includes a ring domain and an acidic domain, both of which are characteristics of certain transcription factors, as well as two putative nuclear localization signals (NLS) that interact with importin-alpha. The normal BRCA1 protein is located in the nucleus of most breast-cell types whereas the BRCA1 protein of breast cancer cells is aberrantly localized in the cytoplasm. This mislocation of the BRCA1 protein in breast cancer cells may be due to defects in the NLS receptor-mediated pathway for the nuclear import of the BRCA1 gene product. Identification of importin-alpha mutations as a cellular protein responsible for the nuclear import of BRCA1 in breast-cancer cell lines and primary breast cancers is the focus of this investigation. METHODS: A series of 15 surgical samples of breast cancer and 3 samples of breast-cancer cell lines (Hs578T, ZR75-1, MCF-7) was assayed for the presence of the deletion mutant in importin-alpha by using both RT-PCR amplification of importin-alpha transcripts and sequencing analysis. RESULTS: Three of the 15 primary breast cancers and 1 of the 3 breast-cancer cell lines showing deletions in importin-alpha transcripts produced two different truncated transcripts. 1208 bp deletions were observed in transcripts from breast cancer (T-1, T-3) and ZR75-1, which is specified by the nucleotide 251-1458 of the transcript. Another transcript encoded by primary breast cancer (T-2) included a 1312 bp deletion in the nucleotide 61-1372 of the transcript. CONCLUSIONS: The deletions eliminated part of the importin-alpha transcript segment encoding the putative NLS-binding domain but not the importin-beta binding domain, suggesting that these deletion mutants could not bind to NLS of the BRCA1 protein. These results suggest that the composite effects of mislocationof the BRCA1 protein by deletion of the NLS-binding domain in importin-alpha may contribute to tumorigenesis in sporadic breast cancer.