• The Korean journal of internal medicine
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• 제34권1호
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• pp.146-155
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• 2019

Background/Aims: Indoxyl sulfate (IS) is a uremic toxin and an important causative factor in the progression of chronic kidney disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to exhibit protective effects in kidney injury. Here, we investigated the effects of paricalcitol treatment on IS-induced renal tubular injury. Methods: The fluorescent dye 2',7'-dichlorofluorescein diacetate was used to measure intracellular reactive oxygen species (ROS) following IS administration in human renal proximal tubular epithelial (HK-2) cells. The effects of IS on cell viability were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and levels of apoptosis-related proteins (Bcl-2-associated protein X [Bax] and B-cell lymphoma 2 [Bcl-2]), nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) p65, and phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) were determined by semiquantitative immunoblotting. The promoter activity of $NF-{\kappa}B$ was measured by luciferase assays and apoptosis was determined by f low cytometry of cells stained with f luorescein isothiocyanate-conjugated Annexin V protein. Results: IS treatment increased ROS production, decreased cell viability and induced apoptosis in HK-2 cells. IS treatment increased the expression of apoptosis-related protein Bax, decreased Bcl-2 expression, and activated phosphorylation of MAPK, $NF-{\kappa}B$ p65, and Akt. In contrast, paricalcitol treatment decreased Bax expression, increased Bcl-2 expression, and inhibited phosphorylation of MAPK, $NF-{\kappa}B$ p65, and Akt in HK-2 cells. $NF-{\kappa}B$ promoter activity was increased following IS, administration and was counteracted by pretreatment with paricalcitol. Additionally, flow cytometry analysis revealed that IS-induced apoptosis was attenuated by paricalcitol treatment, which resulted in decreased numbers of fluorescein isothiocyanate-conjugated Annexin V positive cells. Conclusions: Treatment with paricalcitol inhibited IS-induced apoptosis by regulating MAPK, $NF-{\kappa}B$, and Akt signaling pathway in HK-2 cells.

• Journal of Ginseng Research
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• 제45권6호
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• pp.734-743
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• 2021

Background: The underlying mechanisms of the potential tumor-suppressive effects of ginsenoside Rh2 are complex. N6-methyladenosine (m6A) RNA methylation is usually dysregulated in cancer. This study explored the regulatory effect of ginsenoside Rh2 on m6A RNA methylation in cancer. Methods: m6A RNA quantification and gene-specific m6A RIP-qPCR assays were applied to assess total and gene-specific m6A RNA levels. Co-immunoprecipitation, fractionation western blotting, and immunofluorescence staining were performed to detect protein interactions and distribution. QRT-PCR, dual-luciferase, and ChIP-qPCR assays were conducted to check the transcriptional regulation. Results: Ginsenoside Rh2 reduces m6A RNA methylation and KIF26B expression in a dose-dependent manner in some cancers. KIF26B interacts with ZC3H13 and CBLL1 in the cytoplasm of cancer cells and enhances their nuclear distribution. KIF26B inhibition reduces m6A RNA methylation level in cancer cells. SRF bound to the KIF26B promoter and activated its transcription. SRF mRNA m6A abundance significantly decreased upon KIF26B silencing. SRF knockdown suppressed cancer cell proliferation and growth both in vitro and in vivo, the effect of which was partly rescued by KIF26B overexpression. Conclusion: ginsenoside Rh2 reduces m6A RNA methylation via downregulating KIF26B expression in some cancer cells. KIF26B elevates m6A RNA methylation via enhancing ZC3H13/CBLL1 nuclear localization. KIF26B-SRF forms a positive feedback loop facilitating tumor growth.

• Journal of Microbiology and Biotechnology
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• 제31권10호
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• pp.1331-1342
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• 2021

In this study, we evaluated the mechanism of long non-coding RNA MIR22 host gene (LncRNA MIR22HG) in osteosarcoma cells. Forty-eight paired osteosarcoma and adjacent tissues samples were collected and the bioinformatic analyses were performed. Target genes and potential binding sites of MIR22HG, microRNA (miR)-629-5p and tet methylcytosine dioxygenase 3 (TET3) were predicted by Starbase and TargetScan V7.2 and confirmed by dual-luciferase reporter assay. Cell Counting Kit-8, colony formation and flow cytometry assays were utilized to determine the viability, proliferation and apoptosis of transfected osteosarcoma cells. Pearson's analysis was introduced for the correlation analysis between MIR22HG and miR-629-5p in osteosarcoma tissue. Relative expressions of MIR22HG, miR-629-5p and TET3 were measured by quantitative real-time polymerase chain reaction or Western blot. MiR-629-5p could competitively bind with and was negatively correlated with MIR22HG, the latter of which was evidenced by the high expression of miR-629-5p and low expression of MIR22HG in osteosarcoma tissues. Overexpressed MIR22HG repressed the viability and proliferation but enhanced apoptosis of osteosarcoma cells, which was reversed by miR-629-5p upregulation. TET3 was the target gene of miR-629-5p, and the promotive effects of upregulated miR-629-5p on the viability and proliferation as well as its repressive effect on apoptosis were abrogated via overexpressed TET3. To sum up, overexpressed MIR22HG inhibits the viability and proliferation of osteosarcoma cells, which was achieved via regulation of the miR-629-5p/TET3 axis.

• 대한한의학방제학회지
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• 제26권4호
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• pp.307-318
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• 2018

Objectives : Endoplasmic reticulum (ER) stress designates cellular responses to the accumulation of misfolded and unfolded proteins in ER, which is related to a variety of liver diseases. Present study investigated the inhibitory effects of Litsea japonica flesh water extract (LJE) aganist ER stress. Methods : After HepG2 cells were pretreated with LJE and subsequently exposed to tunicamycin (Tm) or thapsigargin (Tg), expression of C/EBP homologous protein (CHOP), glucose regulated protein 78 kDa (GRP78), asparagine synthetase (ASNS), and endoplasmic reticulum DnaJ homologue 4 (ERDJ4) were determined by immunoblot and real-time PCR analysis. Three canonical signaling pathways in response to ER stress were examined to explore molecular mechanisms involved. Results : Pretreatment of 1 mg/mL LJE inhibited Tm- or Tg-induced CHOP expression, while L. japonica fruit water extract did not. In addition, LJE decreased the levels of GRP78, ASNS, and ERDJ4 mRNA by Tm. Moreover, phosphorylations of eukaryotic translation initiation factor $2{\alpha}$ and inositol-requiring enzyme 1, expression of nuclear form of activating transcription factor $6{\alpha}$, and transactivation of ER stress response element- and unfolded protein response element-harboring luciferase activities were inhibited by LJE pretreatment. Conclusions : Present results suggest that LJE would be a candidate to prevent or treat ER stress-mediated liver injuries.

• BMB Reports
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• 제52권2호
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• pp.157-162
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• 2019

Our previous study found that two novel cancer-related genes, PRR11 and SKA2, constituted a classic gene pair that was regulated by p53 and NF-Y in lung cancer. However, their role and regulatory mechanism in breast cancer remain elusive. In this study, we found that the expression levels of PRR11 and SKA2 were upregulated and have a negative prognotic value in breast cancer. Loss-of-function experiments showed that RNAi-mediated knockdown of PRR11 and/or SKA2 inhibited proliferation, migration, and invasion of breast cancer cells. Mechanistic experiments revealed that knockdown of PRR11 and/or SKA2 caused dysregulation of several downstream genes, including CDK6, TPM3, and USP12, etc. Luciferase reporter assays demonstrated that wild type p53 significantly repressed the PRR11-SKA2 bidirectional promoter activity, but not NF-Y. Interestingly, NF-Y was only essential for and correlated with the expression of PRR11, but not SKA2. Consistently, adriamycin-induced (ADR) activation of endogenous p53 also caused significant repression of the PRR11 and SKA2 gene pair expression. Notably, breast cancer patients with lower expression levels of either PRR11 or SKA2, along with wild type p53, exhibited better disease-free survival compared to others with p53 mutations and/or higher expression levels of either PRR11 or SKA2. Collectively, our study indicates that the PRR11 and SKA2 transcription unit might be an oncogenic contributor and might serve as a novel diagnostic and therapeutic target in breast cancer.

• Journal of Microbiology and Biotechnology
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• 제29권2호
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• pp.321-329
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• 2019

Hair loss, also known as alopecia, is a common dermatological condition of psychosocial significance; development of therapeutic candidates for the treatment of this condition is, hence, important. Silibinin, a secondary metabolite from Silybum marianum, is an effective antioxidant that also prevents various cutaneous problems. In this study, we have investigated the effect of silibinin on hair induction using three-dimensional (3D) cultured, human dermal papilla (DP) spheroids. Silibinin was found to significantly increase viability through AKT serine/threonine kinase (AKT) activation in 3D DP spheroids. This was correlated with an increase in the diameter of the 3D DP spheroids. The activation of the wingless and INT-1 (Wnt)/${\beta}$-catenin signaling pathway, which is associated with hair growth induction in the DP, was evaluated using the T cell-specific transcription factor and lymphoid enhancer-binding factor (TCF/LEF) transcription factor reporter assay; results indicated significantly increased luciferase activity. In addition, we were able to demonstrate increased expression of the target genes, WNT5a and LEF1, using quantitative real-time PCR assay. Lastly, significantly elevated expression of signature genes associated with hair induction was demonstrated in the 3D DP spheroids treated with silibinin. These results suggest that silibinin promotes proliferation and hair induction through the AKT and Wnt/${\beta}$-catenin signaling pathways in 3D DP spheroids. Silibinin can be a potential candidate to promote hair proliferation.

• Journal of Ginseng Research
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• 제43권3호
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• pp.452-459
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• 2019

Background: Ginsenoside compound K(C-K), a major metabolite of ginsenoside, exhibits anticancer activity in various cancer cells and animal models. A cell signaling study has shown that C-K inhibited nuclear factor-kappa B ($NF-{\kappa}B$) pathway in human astroglial cells and liver cancer cells. However, the molecular targets of C-K and the initiating events were not elucidated. Methods: Interaction between C-K and Annexin A2 was determined by molecular docking and thermal shift assay. HepG2 cells were treated with C-K, followed by a luciferase reporter assay for $NF-{\kappa}B$, immunofluorescence imaging for the subcellular localization of Annexin A2 and $NF-{\kappa}B$ p50 subunit, coimmunoprecipitation of Annexin A2 and $NF-{\kappa}B$ p50 subunit, and both cell viability assay and plate clone formation assay to determine the cell viability. Results: Both molecular docking and thermal shift assay positively confirmed the interaction between Annexin A2 and C-K. This interaction prevented the interaction between Annexin A2 and $NF-{\kappa}B$ p50 subunit and their nuclear colocalization, which attenuated the activation of $NF-{\kappa}B$ and the expression of its downstream genes, followed by the activation of caspase 9 and 3. In addition, the overexpression of Annexin A2-K320A, a C-K binding-deficient mutant of Annexin A2, rendered cells to resist C-K treatment, indicating that C-K exerts its cytotoxic activity mainly by targeting Annexin A2. Conclusion: This study for the first time revealed a cellular target of C-K and the molecular mechanism for its anticancer activity.

• 동의생리병리학회지
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• 제33권4호
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• pp.191-197
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• 2019

This study aims to evaluate the effects of the root bark of Morus alba (RMA) on the regulation of the Hippo-YAP pathway. Hippo-YAP signaling is a critical regulator in controlling organ size and tissue homeostasis. Hippo, the serine/threonine kinase phosphorylate the LATS. Phosphorylated LATS then phosphorylates and inactivates the YAP and TAZ, which are two closely related transcriptional co-activator. Here we report RMA activates the Hippo signaling, thereby inhibits the YAP/TAZ activity. First, we examine the cytotoxic effects of RMA by MTT assay. RMA was cytotoxic at concentrations higher than $50{\mu}g/ml$ in HEK293A cells. The reporter gene assay was performed to measure the activity of TEAD, a key transcription factor that controls cell growth and proliferation. RMA significantly suppressed the luciferase activity. By phos-taq gel shift assay, and western blotting, we showed that RMA enhanced the phosphorylation of YAP in wild type cells, but not in LATS1/2 knock out cells, which means RMA activates classical Hippo pathway. RMA induced the cytoplasmic sequestration of YAP. RMA also suppressed the mRNA expression of CTGF and CYR61; the two major YAP dependent genes. Taken together, RMA is considered to be a good candidate for proliferative disease such as cancer, by facilitating cell death through activating the Hippo signaling pathway.

• BMB Reports
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• 제52권9호
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• pp.572-576
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• 2019

Bisphosphonates are the mainstay of therapy worldwide for osteoporosis. However, bisphosphonates also have limitations. The objective of this study was to determine the role of miR-101-3p/Rap1b signal pathway in osteoclast differentiation after treatment with bisphosphonates. Our results revealed that miR-101-3p was an important regulator in bisphosphonates treated-osteoclasts. When miR-101-3p was down-regulated in bone marrow-derived macrophage-like cells (BMMs), the development of mature osteoclasts was promoted, and vice versa. However, alendronate decreased multinucleated cell number regardless of whether miR-101-3p was knocked down or over-expressed. TRAP activity assay confirmed the above results. Luciferase assay indicated that miR-101-3p was a negative regulator of Rap1b. Western blot analysis revealed that protein expression level of Rap1b in BMMs transfected with OV-miR-101-3p was lower than that in BMMs transfected with an empty vector. Rap1b overexpression increased TRAP-positive multinucleated cells, while Rap1b inhibition decreased the cell numbers. In vivo data showed that miR-101-3p inhibited osteoclast differentiation in ovariectomized mice while overexpressed of Rap1b blocked the differentiation. Taken together, our data demonstrate that miR-101-3p/Rap1b signal pathway plays a key role in osteoclast differentiation after treatment with bisphosphonates.

• BMB Reports
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• 제52권9호
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• pp.548-553
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• 2019

Ets-1 is a prototype of the ETS protein family. Members of the ETS protein family contain a unique ETS domain. Ets-1 is associated with cancer progression and metastasis in many types of cancer. Many studies have shown a link between elevated expression of Ets-1 in cancer biopsies and poor survival. CCR7 is a chemokine that binds to specific ligand CCL21/CCL19. CCR7 expression is associated with tumor metastasis and infiltration into lymph nodes. The objective of this study was to test whether Ets-1 could regulate CCR7 expression and enhance tumor metastasis. Our data showed that CCR7 expression was downregulated in Ets-1-deficient T cells upon T-cell stimulation. Overexpression of Ets-1 increased CCR7 expression in breast cancer cell lines. In contrast, knockdown of Ets-1 reduced CCR7 expression. Ets-1 could directly bind to CCR7 promoter and mediate CCR7 expression in luciferase reporter assays and chromatin immunoprecipitation assays. Transactivation activity of Ets-1 was independent of the Pointed domain of Ets-1. Ets-1 could also enhance $NF-{\kappa}B$ and CBP transactivation of CCR7 promoter. Our results also showed that Ets-1 could modulate cancer cell transmigration by altering CCR7 expression in transwell assay and wound healing assay. Taken together, our data suggest that Ets-1 can enhance CCR7 expression and contribute to tumor cell migration.