• BMB Reports
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• 제49권1호
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• pp.51-56
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• 2016

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine protein kinase that is known to mediate cancer cell death. Here, we show that B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, is regulated by GSK-3β and that GSK-3β-mediated regulation of Bcl-2 is crucial for mitochondrial-dependent cell death in paclitaxel-stimulated cells. We demonstrate that MCF7 GSK-3β siRNA cells are more sensitive to cell death than MCF7 GFP control cells and that in the absence of GSK-3β, Bcl-2 levels are reduced, a result enhanced by paclitaxel. Paclitaxel-induced JNK (c-Jun N-terminal kinase) activation is critical for Bcl-2 modulation. In the absence of GSK-3β, Bcl-2 was unstable in an ubiquitination-dependent manner in both basal- and paclitaxel-treated cells. Furthermore, we demonstrate that GSK-3β-mediated regulation of Bcl-2 influences cytochrome C release and mitochondrial membrane potential. Taken together, our data suggest that GSK-3β-dependent regulation of Bcl-2 is crucial for mitochondria-dependent cell death in paclitaxel-mediated breast cancer therapy. [BMB Reports 2016; 49(1): 51-56]

• Archives of Pharmacal Research
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• 제29권5호
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• pp.405-411
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• 2006

Biphenyl dimethyl dicarboxylate (DDB) is a hepatoprotectant, which is used as an adjuvant agent in a treatment for chronic hepatitis. Amantadine is an antiviral agent, which is utilized primarily in the treatment of influenza, but also, occasionally in the treatment of hepatitis C. In a previous study, we reported that DDB, coupled with amantadine, would exert an anti-HBV effect, via the induction of interferon-inducible gene expression in the HepG2 2.2.15 cell line. The primary objective of the present study was to determine whether or not DDB and/or amantadine exhibit anti-HBV properties, and what mechanisms of action might be involved in such properties. In our study, we were able to determine that DDB stimulates Jak/Stat signaling, and induces the expression of interferon alpha $(IFN-\alpha)$ stimulated genes, most notably 6-16 and ISG12. In addition, the antiviral effectors induced by $IFN-\alpha$, PKR, OAS, and MxA, were regulated in the presence of DDB at its optimal concentration $(250{\mu}g/mL)$, to a degree commensurate with the degree of induction associated with the $IFN-\alpha$ treated group. Finally, we determined that the replication of pregenomic RNA and HBeAg was inhibited by DDB treatment, and this inhibition was maximized when coupled with the administration of amantadine $(25{\mu}g/mL)$. In conclusion, the results of this study demonstrated clearly that DDB, as well as the combination of DDB/amantadine, directly inhibited $IFN-\alpha$ signaling-mediated replication of HBV in infected hepatocytes, and thus may represent a novel treatment for chronic hepatitis B, which would be characterized principally by its improved safety over other treatment strategies.

• Molecules and Cells
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• 제21권1호
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• pp.104-111
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• 2006

Gene therapy with nonviral vectors using the suicide gene/prodrug activating system of herpes simplex virus type-1 thymidine kinase (HSV1-TK)/ganciclovir (GCV) is inefficient in killing malignant tumor cells due to two major factors: (a) an unsatisfactory bystander effect; (b) short-lived expression of the protein. To study the capacity of the protein transduction domain (PTD) of HIV-1 TAT protein to enhance HSV1-TK/GCV cancer gene therapy, we constructed three fusion proteins TAT-TK, TK-TAT and TK. TAT-TK retained as much enzyme activity as TK, whereas that of TK-TAT was much lower. TAT-TK can enter HepG2 cells and much of it is translocated to the nucleus. The transduced HepG2 cells are killed by exogenously added GCV and have bystander effects on untransduced HepG2 cells. Most importantly, the introduced recombinant protein is stable and remains functional for several days at least, probably because nuclear localization protects it from the cytoplasmic degradation machinery and provides access to the nuclear transcription machinery. Our results indicate that TAT fusion proteins traffic intercellularly and have enhanced stability and prodrug cell killing activity. We conclude that TAT has potential for enhancing enzyme prodrug treatment of liver cancers.

• Molecules and Cells
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• 제44권8호
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• pp.580-590
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• 2021

Patients with severe asthma have unmet clinical needs for effective and safe therapies. One possibility may be mesenchymal stem cell (MSC) therapy, which can improve asthma in murine models. However, it remains unclear how MSCs exert their beneficial effects in asthma. Here, we examined the effect of human umbilical cord blood-derived MSCs (hUC-MSC) on two mouse models of severe asthma, namely, Alternaria alternata-induced and house dust mite (HDM)/diesel exhaust particle (DEP)-induced asthma. hUC-MSC treatment attenuated lung type 2 (Th2 and type 2 innate lymphoid cell) inflammation in both models. However, these effects were only observed with particular treatment routes and timings. In vitro co-culture showed that hUC-MSC directly downregulated the interleukin (IL)-5 and IL-13 production of differentiated mouse Th2 cells and peripheral blood mononuclear cells from asthma patients. Thus, these results showed that hUC-MSC treatment can ameliorate asthma by suppressing the asthmogenic cytokine production of effector cells. However, the successful clinical application of MSCs in the future is likely to require careful optimization of the route, dosage, and timing.

• Molecules and Cells
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• 제45권12호
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• pp.935-949
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• 2022

Liver cancer has a high prevalence, with majority of the cases presenting as hepatocellular carcinoma (HCC). The prognosis of metastatic HCC has hardly improved over the past decade, highlighting the necessity for liver cancer research. Studies have reported the ability of the KiSS1 gene to inhibit the growth or metastasis of liver cancer, but contradictory research results are also emerging. We, therefore, sought to investigate the effects of KiSS1 on growth and migration in human HCC cells. HepG2 human HCC cells were infected with lentivirus particles containing KiSS1. The overexpression of KiSS1 resulted in an increased proliferation rate of HCC cells. Quantitative polymerase chain reaction and immunoblotting revealed increased Akt activity, and downregulation of the G1/S phase cell cycle inhibitors. A significant increase in tumor spheroid formation with upregulation of β-catenin and CD133 was also observed. KiSS1 overexpression promoted the migratory, invasive ability, and metastatic capacity of the hepatocarcinoma cell line, and these effects were associated with changes in the expressions of epithelial mesenchymal transition (EMT)- related genes such as E-cadherin, N-cadherin, and slug. KiSS1 overexpression also resulted in dramatically increased tumor growth in the xenograft mouse model, and upregulation of proliferating cell nuclear antigen (PCNA) and Ki-67 in the HCC tumors. Furthermore, KiSS1 increased the angiogenic capacity by upregulation of the vascular endothelial growth factor A (VEGF-A) and CD31. Based on these observations, we infer that KiSS1 not only induces HCC proliferation, but also increases the metastatic potential by increasing the migratory ability and angiogenic capacity.

• IMMUNE NETWORK
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• 제22권6호
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• pp.50.1-50.19
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• 2022

Autoreactive B cells are not entirely deleted, but some remain as immunocompetent or anergic B cells. Although the persistence of autoreactive B cells as anergic cells has been shown in transgenic mouse models with the expression of B cell receptor (BCR) reactive to engineered self-antigen, the characterization of naturally occurring anergic B cells is important to identify them and understand their contribution to immune regulation or autoimmune diseases. We report here that a low-level expression of CD138 in the splenic B cells marks naturally arising anergic B cells, not plasma cells. The CD138^int B cells consisted of IgM^lowIgD^high follicular (FO) B cells and transitional 3 B cells in homeostatic conditions. The CD138^int FO B cells showed an anergic gene expression profile shared with that of monoclonal anergic B cells expressing engineered BCRs and the gene expression profile was different from those of plasma cells, age-associated B cells, or germinal center B cells. The anergic state of the CD138^int FO B cells was confirmed by attenuated Ca²⁺ response and failure to upregulate CD69 upon BCR engagement with anti-IgM, anti-IgD, anti-Igκ, or anti-IgG. The BCR repertoire of the CD138^int FO B cells was distinct from that of the CD138^- FO B cells and included some class-switched B cells with low-level somatic mutations. These findings demonstrate the presence of polyclonal anergic B cells in the normal mice that are characterized by low-level expression of CD138, IgM downregulation, reduced Ca²⁺ and CD69 responses upon BCR engagement, and distinct BCR repertoire.

• IMMUNE NETWORK
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• 제22권6호
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• pp.51.1-51.20
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• 2022

Trypanosoma cruzi, the etiological agent of Chagas disease, is an intracellular protozoan parasite, which is now present in most industrialized countries. About 40% of T. cruzi infected individuals will develop severe, incurable cardiovascular, gastrointestinal, or neurological disorders. The molecular mechanisms by which T. cruzi induces cardiopathogenesis remain to be determined. Previous studies showed that increased IL-6 expression in T. cruzi patients was associated with disease severity. IL-6 signaling was suggested to induce pro-inflammatory and pro-fibrotic responses, however, the role of this pathway during early infection remains to be elucidated. We reported that T. cruzi can dysregulate the expression of host PIWI-interacting RNAs (piRNAs) during early infection. Here, we aim to evaluate the dysregulation of IL-6 signaling and the piRNAs computationally predicted to target IL-6 molecules during early T. cruzi infection of primary human cardiac fibroblasts (PHCF). Using in silico analysis, we predict that piR_004506, piR_001356, and piR_017716 target IL6 and SOCS3 genes, respectively. We validated the piRNAs and target gene expression in T. cruzi challenged PHCF. Secreted IL-6, soluble gp-130, and sIL-6R in condition media were measured using a cytokine array and western blot analysis was used to measure pathway activation. We created a network of piRNAs, target genes, and genes within one degree of biological interaction. Our analysis revealed an inverse relationship between piRNA expression and the target transcripts during early infection, denoting the IL-6 pathway targeting piRNAs can be developed as potential therapeutics to mitigate T. cruzi cardiomyopathies.

• IMMUNE NETWORK
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• 제21권5호
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• pp.37.1-37.17
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• 2021

Hepatitis B virus X (HBx) protein has been reported as a key protein regulating the pathogenesis of HBV-induced hepatocellular carcinoma (HCC). Recent evidence has shown that HBx is implicated in the activation of autophagy in hepatic cells. Nevertheless, the precise molecular and cellular mechanism by which HBx induces autophagy is still controversial. Herein, we investigated the molecular and cellular mechanism by which HBx is involved in the TRAF6-BECN1-Bcl-2 signaling for the regulation of autophagy in response to TLR4 stimulation, therefore influencing the HCC progression. HBx interacts with BECN1 (Beclin 1) and inhibits the association of the BECN1-Bcl-2 complex, which is known to prevent the assembly of the pre-autophagosomal structure. Furthermore, HBx enhances the interaction between VPS34 and TRAF6-BECN1 complex, increases the ubiquitination of BECN1, and subsequently enhances autophagy induction in response to LPS stimulation. To verify the functional role of HBx in liver cancer progression, we utilized different HCC cell lines, HepG2, SK-Hep-1, and SNU-761. HBx-expressing HepG2 cells exhibited enhanced cell migration, invasion, and cell mobility in response to LPS stimulation compared to those of control HepG2 cells. These results were consistently observed in HBx-expressed SK-Hep-1 and HBx-expressed SNU-761 cells. Taken together, our findings suggest that HBx positively regulates the induction of autophagy through the inhibition of the BECN1-Bcl-2 complex and enhancement of the TRAF6-BECN1-VPS34 complex, leading to enhance liver cancer migration and invasion.

• IMMUNE NETWORK
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• 제8권1호
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• pp.21-28
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• 2008

Background: A co-inhibitory molecule, B7-H4, is believed to negatively regulate T cell immunity by suppressing T cell proliferation and inhibiting cytokine production. However, the mechanism behind B7-H4-mediated tolerance remains unclear. Methods: Balb/c $(H-2^d)$ mice were fed with dendritic cell line, DC2.4 $(H-2^d)$ every day for 10 days. Meantime, mice were hydrodynamically injected with recombinant plasmid expressing B7-H4 fusion protein (B7-H4.hFc) or hFc via tail vein. One day after last feeding, mice were immunized with allogeneic B6 spleen cells. 14 days following immunization, mice were challenged with B6 spleen cells to ear back and the ear swelling was determined the next day. Subsequently, a mixed lymphocyte reaction (MLR) was also performed and cytokines profiles from the reaction were examined by sandwich ELISA. Frequency of immunosuppressive cell population was assayed with flow cytometry and mRNA for FoxP3 was determined by RT-PCR. Results: Tolerant mice given plasmid expressing B7-H4.hFc showed a significant reduction in ear swelling compared to control mice. In addition, T cells from mice given B7-H4.hFc plasmid revealed a significant hyporesponsiveness of T cells against allogeneic spleen cells and showed a significant decrease in Th1 and Th2 cytokines such as IFN-${\gamma}$, IL-5, and TNF-${\alpha}$. Interestingly, flow cytometric analysis showed that the frequency of CD4+CD25+FoxP3+ Tregs in spleen was increased in tolerant mice given recombinant B7-H4.hFc plasmid compared to control group. Conclusion: Our results demonstrate that B7-H4 synergistically potentiates oral tolerance induced by allogeneic cells by increasing the frequency of FoxP3+ CD4+CD25+ Treg and reducing Th1 and Th2 cytokine production.

• Journal of Microbiology and Biotechnology
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• 제11권4호
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• pp.677-684
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• 2001

To isolate mutanse-producing bacteria, soil samples were collected from several areas in chonnam Province, South Korea. A total of 70 strains of actinomycetes were isolated from the soil samples. All isolated actinomycetes were inoculated on mutanase screening media to identify new bacterial strains producing mutanase activity. One strain in particular exhibited a strong mutanase-producing activity, and was identified as Microbispora rosea based on its morphological, cultural, and physiological characteristics, and also by 16S rDNA sequences.