• Herbal Formula Science
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• v.27 no.1
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• pp.17-29
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• 2019

Inflammatory and oxidative stimuli play a critical role not only in the process of transforming normal cells into cancer cells, but also in the proliferation process of cancer cells. Sipyukmiryukieum (SYMRKU), a traditional Korean herb-combined remedy, is composed of 16 kinds of herbal medicines, which were recorded for "Ongjeo" treatment in "Dongeuibogam". In this study, we investigated the inhibitory effect of SYMRKU against inflammatory and oxidative responses in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Our results showed that SYMRKU significantly inhibited LPS-induced secretion of pro-inflammatory mediators including nitric oxide (NO) and prostaglandin $E_2$ without showing any significant cytotoxicity. Consistent with these results, SYMRKU down-regulated LPS-induced expression of their regulatory enzymes such as inducible NO synthase and cyclooxygenase-2. SYMRKU also inhibited LPS-induced production and expression of pro-inflammatory cytokines such as tumor necrosis factor-${\alpha}$, interleukin (IL)-$1{\beta}$ and IL-6. In addition, SYMRKU significantly reduced the production of reactive oxygen species by LPS and showed a strong, which was associated with induction of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 expression. Although further studies are needed to fully understand the anti-inflammatory effects associated with the antioxidant capacity of SYMRKU, the findings of the current study suggest that SYMRKU may have potential benefits by inhibiting the onset and/or treatment of inflammatory and/or oxidative diseases.

• Journal of Korean Neurosurgical Society
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• v.64 no.1
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• pp.100-109
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• 2021

Objective : Diffuse astrocytic tumour (DAT) is a diffuse infiltrative astrocytoma tumour accompanied by molecular parameters such as the presence or absence of isocitrate dehydrogenase (IDH) gene mutations. Ki-67 is a marker for DAT proliferation, while programmed death ligand 1 (PD-L1) indicates an immune evasion mechanism. This study aimed to analyze the correlation among mutant IDH1 R132H, Ki-67, and PD-L1 immunoexpression in the DAT. Methods : A cross-sectional study was carried out on 30 paraffin blocks of DAT cases. Paraffin block samples consist of grade II (n=14), grade III (n=8), and grade IV (n=8). In this study, the immunohistochemistry-staining of mutant IDH1 R132H, Ki-67, and PD-L1 were carried out to determine the frequency of DAT with IDH1 mutations. Results : Our study shown the frequency of IDH1 mutations in grade II 50.0% (7/14), grade III 37.5% (3/8), and grade IV 12.5% (1/8). Our study also showed a difference in Ki-67 and PD-L1 expression between each the degree of DAT histopathology (p=0.0001 and p=0.002, respectively). There was an association between both mutant IDH1 R132H, and Ki-67 with PD-L1 expression in DAT (p=0.0087 and p=0.0049, respectively). Conclusion : DAT with the mutant IDH1 is frequently observed in grade II and small number of grade III. The expression of wild type IDH1, Ki-67, and PD-L1 were found to be higher in high grade DAT (grade III and grade IV). There is a correlation between each of mutant IDH1 status and Ki-67 with PD-L1 expression in DAT.

• Journal of Ginseng Research
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• v.46 no.3
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• pp.418-425
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• 2022

Background: Sorafenib is effective in treating hepatoma, but most patients develop resistance to it. STAT3 signaling has been implicated in sorafenib resistance. Artesunate (ART) and 20(R)-ginsenoside Rg3 (Rg3) have anti-hepatoma effects and can inhibit STAT3 signaling in cancer cells. This study aimed to evaluate the effects of Rg3 in combination with ART (Rg3-plus-ART) in overcoming sorafenib resistance, and to examine the involvement of STAT3 signaling in these effects. Methods: Sorafenib-resistant HepG2 cells (HepG2-SR) were used to evaluate the in vitro anti-hepatoma effects of Rg3-plus-ART. A HepG2-SR hepatoma-bearing BALB/c-nu/nu mouse model was used to assess the in vivo anti-hepatoma effects of Rg3-plus-ART. CCK-8 assays and Annexin V-FITC/PI double staining were used to examine cell proliferation and apoptosis, respectively. Immunoblotting was employed to examine protein levels. ROS generation was examined by measuring DCF-DA fluorescence. Results: Rg3-plus-ART synergistically reduced viability of, and evoked apoptosis in HepG2-SR cells, and suppressed HepG2-SR tumor growth in mice. Mechanistic studies revealed that Rg3-plus-ART inhibited activation/phosphorylation of Src and STAT3 in HepG2-SR cultures and tumors. The combination also decreased the STAT3 nuclear level and induced ROS production in HepG2-SR cultures. Furthermore, overactivation of STAT3 or removal of ROS diminished the anti-proliferative effects of Rg3-plus-ART, and removal of ROS diminished Rg3-plus-ART's inhibitory effects on STAT3 activation in HepG2-SR cells. Conclusions: Rg3-plus-ART overcomes sorafenib resistance in experimental models, and inhibition of Src/STAT3 signaling and modulation of ROS/STAT3 signaling contribute to the underlying mechanisms. This study provides a pharmacological basis for developing Rg3-plus-ART into a novel modality for treating sorafenib-resistant hepatoma.

• Biomolecules & Therapeutics
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• v.30 no.6
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• pp.553-561
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• 2022

Chronic myeloid leukemia (CML) is a slowly progressing hematopoietic cell disorder. Sphingosine kinase 1 (SPHK1) plays established roles in tumor initiation, progression, and chemotherapy resistance in a wide range of cancers, including leukemia. However, small-molecule inhibitors targeting SPHK1 in CML still need to be developed. This study revealed the role of SPHK1 in CML and investigated the potential anti-leukemic activity of hirsuteine (HST), an indole alkaloid obtained from the oriental plant Uncaria rhynchophylla, in CML cells. These results suggest that SPHK1 is highly expressed in CML cells and that overexpression of SPHK1 represents poor clinical outcomes in CML patients. HST exposure led to G2/M phase arrest, cellular apoptosis, and downregulation of Cyclin B1 and CDC2 and cleavage of Caspase 3 and PARP in CML cells. HST shifted sphingolipid rheostat from sphingosine 1-phosphate (S1P) towards the ceramide coupled with a marked inhibition of SPHK1. Mechanistically, HST significantly blocked SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt pathways. In addition, HST can be docked with residues of SPHK1 and shifts the SPHK1 melting curve, indicating the potential protein-ligand interactions between SPHK1 and HST in both CML cells. SPHK1 overexpression impaired apoptosis and proliferation of CML cells induced by HST alone. These results suggest that HST, which may serve as a novel and specific SPHK1 inhibitor, exerts anti-leukemic activity by inhibiting the SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt pathways in CML cells, thus conferring HST as a promising anti-leukemic drug for CML therapy in the future.

• Nutrition Research and Practice
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• v.16 no.6
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• pp.685-699
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• 2022

BACKGROUND/OBJECTIVES: Platycodon grandiflorum (PG) has long been known as a medicinal herb effective in various diseases, including bronchitis and asthma, but is still more widely used for food. Fermentation methods are being applied to increase the pharmacological composition of PG extracts and commercialize them with high added value. This study examines the hydrolyzed and fermented PG extract (HFPGE) fermented with Lactobacillus casei in RAW 264.7 cells, and investigates the effect of amplifying the immune and the probable molecular mechanism. MATERIALS/METHODS: HFPGE's total phenolic, flavonoid, saponin, and platycodin D contents were analyzed by colorimetric analysis or high-performance liquid chromatography. Cell viability was measured by the MTT assay. Phagocytic activity was analyzed by a phagocytosis assay kit, nitric oxide (NO) production by a Griess reagent system, and cytokines by enzyme-linked immunosorbent assay kits. The mRNA expressions of inducible nitric oxide synthase (iNOS) and cytokines were analyzed by reverse transcription-polymerase chain reaction, whereas MAPK and nuclear factor (NF)-κB activation were analyzed by Western blots. RESULTS: Compared to PGE, HFPGE was determined to contain 13.76 times and 6.69 times higher contents of crude saponin and platycodin D, respectively. HFPGE promoted cell proliferation and phagocytosis in RAW 264.7 cells and regulated the NO production and iNOS expression. Treatment with HFPGE also resulted in increased production of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, C-X-C motif chemokine ligand10, granulocyte-colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-1, and the mRNA expressions of these cytokines. HFPGE also resulted in significantly increasing the phosphorylation of NF-κB p65, extracellular signal-regulated kinase, and c-Jun N-terminal kinase. CONCLUSIONS: Taken together, our results imply that fermentation and hydrolysis result in the extraction of more active ingredients of PG. Furthermore, we determined that HFPGE exerts immunostimulatory activity via the MAPK and NF-κB signaling pathways.

• Proceedings of the Korean Society of Toxicology Conference
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• 2000.11a
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• pp.45-67
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• 2000

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a mutagenic and carcinogenic heterocyclic amino found in cooked meat. The in vivo mutagenicity and hepatocarcinogenicity of MeIQx were examined in mice harboring the lacZ mutation reporter gene ($Muta^{TM}$ Mice) and bitransgenic mice over-expressing the c-myc oncogene. C57B1/$\lambda$lacZ and bitransgenic c-myc (albumin promoter)/$\lambda$lacZ mice were bred and weaned onto an AIN-76 based diet containing $0.06\%$ (w/w) MeIQx or onto control diet. After 30 weeks on diet, only male bitransgenic mice on MeIQx developed hepatocellular carcinoma ($100\%$ incidence) indicating that there was synergism between c-myc over-expression and MeIQx. By 40 weeks, hepatic tumor incidence was $100\%$ ($17\%$) and $44\%$ ($0\%$) in male c-myc/$\lambda$lacZ and C57B1/$\lambda$lacZ mice given MeIQx (or control) diet, respectively, indicating that either MeIQx or c-myc over-expression alone eventually induced hepatic tumors. At either time point, mutant frequency in the lacZ gene was at least 40-fold higher in MeIQx-treated mice than in control mice of either strain. These findings suggest that MeIQx-induced hepatocarcinogenesis is associated with MeIQx-induced mutations. Elevated mutant frequency in MeIQx-treated mice also occurred concomitant with the formation of MeIQx-guanine adducts as detected by the $^{32}P$-postlabeling assay. Irrespective of strain or diet, sequence analysis of the lacZ mutants from male mouse liver showed that the principal sequence alteration was a single guanine-base substitution. Adenine mutations, however, were detected only in animals on control diet. MeIQx-fed mice harboring the c-myc oncogene showed a l.4-2.6-fold higher mutant frequency in the lacZ gene than mice not carrying the transgene. Although there was a trend toward higher adduct levels in c-myc mice, MeIQx-DNA adduct levels were not significantly different between c-myc/$\lambda$lacZ and C57B1/$\lambda$lacZ mice after 30 weeks on diet. Thus, it appeared that factors in addition to MeIQx-DNA adduct levels, such as the enhance rate of proliferation associated with c-myc over-expression, may have accounted for a higher mutant frequency in c-myc mice. In the control diet groups, the lacZ mutant frequency was significantly higher in c-myc/$\lambda$lacZ mice than in 057B1/$\lambda$1acZ mice. The findings are consistent with the notion that c-myc over-expression is associated with an increase in mutagenesis. The mechanism for the synergistic effects of c-myc over-expression on MeIQx hepatocarcinogenicity appears to involve an enhancement of MeIQx-induced mutations.

• The Korea Journal of Herbology
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• v.24 no.3
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• pp.161-167
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• 2009

Objectives : Maekmoondong-tang (MMDT), a Korean herbal medicine, has been used to treat severe dry cough in patients with bronchitis and pharyngitis. MMDT has been reported to have anti-inflammatory, anti-allergic, immunomodulatory, secretory-modulating, and metabolic regulatory actions. However, there are no evidence in regard to the effects of MMDT on carcinogenesis and metastasis. Here, we investigated the effects of 70% ethanol extract of MMDT on cell viability, apoptosis, and motility in human hepatocarcinoma HepG2 cells. Methods : Cell viability was measured using the CCK-8 assay, and the apoptosis induction was evaluated by caspase-3 activity. To detect apoptotic features, the cells treated with MMDT were stained with 4'-6-diamidino-2-phenylindole (DAPI). Cell motility was examined by Boyden chamber assay and Real-time Cell Index of Migration assay. Gelatin zymography also performed to measure matrix metalloproteinase (MMP)-2/9 activity. Results : We found that MMDT significantly inhibited cell proliferation and increased caspase-3 activity in a dose-dependent manner in HepG2 cells. Apoptotic features such as chromatin condensation and apoptotic bodies were observed in MMDT-treated cells by DAPI staining. MMDT also suppressed PMA-induced cell motility and activities of MMP-2/9. Conclusions : Our results exhibited that MMDT possess the anti-carcinogenetic and anti-metastatic activities via caspase-3 activation and down-regulation of cell motility and invasion in HepG2 cells. Therefore, these findings suggest that MMDT could be potentially applied to the prevention and treatment of cancer.

• Animal Bioscience
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• v.36 no.6
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• pp.851-860
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• 2023

Objective: This study aims to evaluate the target genes of gga-miR-20a-5p and the regulated immune responses in the chicken macrophage cell line, HD11, by the exosome-mediated delivery of miR-20a-5p. Methods: Exosomes were purified from the chicken macrophage cell line HD11. Then, mimic gga-miR-20p or negative control miRNA were internalized into HD11 exosomes. HD11 cells were transfected with gga-miR-20a-5p or negative control miRNA containing exosomes. After 44 h of transfection, cells were incubated with or without 5 ㎍/mL poly(I:C) for 4 h. Then, expression of target genes and cytokines was evaluated by quantitative realtime polymerase chain reaction. Results: Using a luciferase reporter assay, we identified that gga-miR-20a-5p directly targeted interferon gamma receptor 2 (IFNGR2), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase kinase kinase 5 (MAP3K5), and mitogen-activated protein kinase kinase kinase 14 (MAP3K14). Moreover, the exosome-mediated delivery of gga-miR-20a-5p successfully repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14 in HD11 cells. The expressions of interferon-stimulated genes (MX dynamin like GTPase 1 [MX1], eukaryotic translation initiation factor 2A [EIF2A], and oligoadenylate synthase-like [OASL]) and proinflammatory cytokines (interferon-gamma [IFNG], interleukin-1 beta [IL1B], and tumor necrosis factor-alpha [TNFA]) were also downregulated by exosomal miR-20a-5p. In addition, the proliferation of HD11 cells was increased by exosomal miR-20a-5p. Conclusion: The exosome-mediated delivery of gga-miR-20a-5p regulated immune responses by controlling the MAPK and apoptotic signaling pathways. Furthermore, we expected that exosomal miR-20a-5p could maintain immune homeostasis against highly pathogenic avian influenza virus H5N1 infection by regulating the expression of proinflammatory cytokines and cell death.

• Journal of Ginseng Research
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• v.47 no.4
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• pp.515-523
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• 2023

Background: 20(S)-protopanaxadiol (PPD), one of the main components of ginseng, has anti-inflammatory, anti-estrogenic, and anti-tumor activities. It is known that activated hepatic stellate cells (HSCs) are the primary producers of extracellular matrix (ECM) in the liver, and the Wnt/β-catenin pathway participates in the activation of HSCs. We aimed to explore whether PPD inhibits liver fibrosis is associated with the Wnt/β-catenin pathway inactivation. Methods: The anti-fibrotic roles of PPD were examined both in vitro and in vivo. We also examined the levels of Wnt inhibitory factor 1 (WIF1), DNA methyltransferase 1 (DNMT1) and WIF1 methylation. Results: PPD obviously ameliorated liver fibrosis in carbon tetrachloride (CCl₄)-treated mice and reduced collagen deposition. PPD also suppressed the activation and proliferation of primary HSCs. Notably, PPD inhibited the Wnt/β-catenin pathway, reduced TCF activity, and increased P-β-catenin and GSK-3β levels. Interestingly, WIF1 was found to mediate the inactivation of the Wnt/β-catenin pathway in PPD-treated HSCs. WIF1 silencing suppressed the inhibitory effects of PPD on HSC activation and also restored α-SMA and type I collagen levels. The downregulation of WIF1 expression was associated with the methylation of its promoter. PPD induced WIF1 demethylation and restored WIF1 expression. Further experiments confirmed that DNMT1 overexpression blocked the effects of PPD on WIF1 expression and demethylation and enhanced HSC activation. Conclusion: PPD up-regulates WIF1 levels and impairs Wnt/β-catenin pathway activation via the downregulation of DNMT1-mediated WIF1 methylation, leading to HSC inactivation. Therefore, PPD may be a promising therapeutic drug for patients with liver fibrosis.

• Journal of Microbiology and Biotechnology
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• v.33 no.9
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• pp.1206-1212
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• 2023

The Wnt/β-catenin pathway plays essential roles in regulating various cellular behaviors, including proliferation, survival, and differentiation [1-3]. The intracellular β-catenin level, which is regulated by a proteasomal degradation pathway, is critical to Wnt/β-catenin pathway control [4]. Normally, casein kinase 1 (CK1) and glycogen synthase kinase-3β (GSK-3β), which form a complex with the scaffolding protein Axin and the tumor suppressor protein adenomatous polyposis coli (APC), phosphorylate β-catenin at Ser45, Thr41, Ser37, and Ser33 [5, 6]. Phosphorylated β-catenin is ubiquitinated by the β-transducin repeat-containing protein (β-TrCP), an F-box E3 ubiquitin ligase complex, and ubiquitinated β-catenin is degraded via a proteasome pathway [7, 8]. Colorectal cancer is a significant cause of cancer-related deaths worldwide. Abnormal up-regulation of the Wnt/β-catenin pathway is a major pathological event in intestinal epithelial cells during human colorectal cancer oncogenesis [9]. Genetic mutations in the APC gene are observed in familial adenomatous polyposis coli (FAP) and sporadic colorectal cancers [10]. In addition, mutations in the N-terminal phosphorylation motif of the β-catenin gene were found in patients with colorectal cancer [11]. These mutations cause β-catenin to accumulate in the nucleus, where it forms complexes with transcription factors of the T-cell factor/lymphocyte enhancer factor (TCF/LEF) family to stimulate the expression of β-catenin responsive genes, such as c-Myc and cyclin D1, which leads to colorectal tumorigenesis [12-14]. Therefore, downregulating β-catenin response transcription (CRT) is a potential strategy for preventing and treating colorectal cancer. Plant cytokinins are N⁶-substituted purine derivatives; they promote cell division in plants and regulate developmental pathways. Natural cytokinins are classified as isoprenoid (isopentenyladenine, zeatin, and dihydrozeatin), aromatic (benzyladenine, topolin, and methoxytopolin), or furfural (kinetin and kinetin riboside), depending on their structure [15, 16]. Kinetin riboside was identified in coconut water and is a naturally produced cytokinin that induces apoptosis and exhibits antiproliferative activity in several human cancer cell lines [17]. However, little attention has been paid to kinetin riboside's mode of action. In this study, we show that kinetin riboside exerts its cytotoxic activity against colon cancer cells by suppressing the Wnt/β-catenin pathway and promoting intracellular β-catenin degradation.