• Biomolecules & Therapeutics
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• 제27권6호
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• pp.553-561
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• 2019

Rab25, a member of the Rab11 small GTPase family, is central to achieving cellular polarity in epithelial tissues. Rab25 is highly expressed in epithelial cells of various tissues including breast, vagina, cervix, the gastrointestinal tract, and skin. Rab25 plays key roles in tumorigenesis, mainly by regulating epithelial differentiation and proliferation. However, its role in skin physiology is relatively unknown. In this study, we demonstrated that Rab25 knock-out (KO) mice show a skin barrier dysfunction with high trans-epidermal water loss and low cutaneous hydration. To examine this observation, we investigated the histology and epidermal differentiation markers of the skin in Rab25 KO mice. Rab25 KO increased cell proliferation at the basal layer of epidermis, whereas the supra-basal layer remained unaffected. Ceramide, which is a critical lipid component for skin barrier function, was not altered by Rab25 KO in its distribution or amount, as determined by immunohistochemistry. Notably, levels of epidermal differentiation markers, including loricrin, involucrin, and keratins (5, 14, 1, and 10) increased prominently in Rab25 KO mice. In line with this, depletion of Rab25 with single hairpin RNA increased the expression of differentiation markers in a human keratinocyte cell line, HaCaT. Transcriptomic analysis of the skin revealed increased expression of genes associated with skin development, epidermal development, and keratinocyte differentiation in Rab25 KO mice. Collectively, these results suggested that Rab25 is involved in the regulation of epidermal differentiation and proliferation.

• Biomolecules & Therapeutics
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• 제27권6호
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• pp.570-576
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• 2019

Particulate matter (PM), which refers to the mixture of particles present in the air, can have harmful effects. Damage to cells by PM, including disruption of organelles and proteins, can trigger autophagy, and the relationship between autophagy and PM has been well studied. However, the cellular regulators of PM-induced autophagy have not been well characterized, especially in keratinocytes. The Aryl Hydrocarbon Receptor (AhR) is expressed in the epidermis and is activated by PM. In this study, we investigated the role of the AhR in PM-induced autophagy in HaCaT cells. Our results showed that PM led to AhR activation in keratinocytes. Activation of the AhR-target gene CYP1A1 by PM was reduced by co-treatment with ${\alpha}$-naphthoflavone (${\alpha}-NF$), an AhR inhibitor. We also evaluated activation of the autophagy pathway in PM-treated keratinocytes. In HaCaT cells, treatment with PM treatment led to the induction of microtubules-associated proteins light chain 3 (LC3) and p62/SQSTM1, which are essential components of the autophagy pathway. To study the role of the AhR in mediating PM-induced autophagy, we treated cells with ${\alpha}-NF$ or used an siRNA against AhR. Expression of LC3-II induced by PM was decreased in a dose dependent manner by ${\alpha}-NF$. Furthermore, knockdown of AhR with siAhR diminished PM-induced expression of LC3-II and p62. Together, these results suggest that inhibition of the AhR decreases PM-induced autophagy. We confirmed these results using the autophagy-inhibitors BAF and 3-MA. Taken together, our results indicate that exposure to PM induces autophagy via the AhR in HaCaT keratinocytes.

• Biomolecules & Therapeutics
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• 제27권6호
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• pp.591-602
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• 2019

Human breast cancer cell line, MDA-MB-231, is highly invasive and aggressive, compared to less invasive cell line, MCF-7. To explore the genes that might influence the malignancy of MDA-MB-231, DNA microarray analysis was performed. The results showed that G0/G1 switch 2 (G0S2) was one of the most highly expressed genes among the genes upregulated in MDA-MB-231. Although G0S2 acts as a direct inhibitor of adipose triglyceride lipase, action of G0S2 in cancer progression is not yet understood. To investigate whether G0S2 affects invasiveness of MDA-MB-231 cells, G0S2 expression was inhibited using siRNA, which led to decreased cell proliferation, migration, and invasion of MDA-MB-231 cells. Consequently, G0S2 inhibition inactivated integrin-regulated FAK-Src signaling, which promoted Hippo signaling and inactivated ERK1/2 signaling. In addition, G0S2 downregulation decreased ${\beta}$-catenin expression, while E-cadherin expression was increased. It was demonstrated for the first time that G0S2 mediates the Hippo pathway and induces epithelial to mesenchymal transition (EMT). Taken together, our results suggest that G0S2 is a major factor contributing to cell survival and metastasis of MDA-MB-231 cells.

• Biomolecules & Therapeutics
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• 제27권3호
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• pp.290-301
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• 2019

Paeonol has neuroprotective function, which could be useful for improving central nervous system disorder. The purpose of this study was to characterize the functional mechanism involved in brain transport of paeonol through blood-brain barrier (BBB). Brain transport of paeonol was characterized by internal carotid artery perfusion (ICAP), carotid artery single injection technique (brain uptake index, BUI) and intravenous (IV) injection technique in vivo. The transport mechanism of paeonol was examined using conditionally immortalized rat brain capillary endothelial cell line (TR-BBB) as an in vitro model of BBB. Brain volume of distribution (VD) of [$^3H$]paeonol in rat brain was about 6-fold higher than that of [$^{14}C$]sucrose, the vascular space marker of BBB. The uptake of [$^3H$]paeonol was concentration-dependent. Brain volume of distribution of paeonol and BUI as in vivo and inhibition of analog as in vitro studies presented significant reduction effect in the presence of unlabeled lipophilic compounds such as paeonol, imperatorin, diphenhydramine, pyrilamine, tramadol and ALC during the uptake of [$^3H$]paeonol. In addition, the uptake significantly decreased and increased at the acidic and alkaline pH in both extracellular and intracellular study, respectively. In the presence of metabolic inhibitor, the uptake reduced significantly but not affected by sodium free or membrane potential disruption. Similarly, paeonol uptake was not affected on OCTN2 or rPMAT siRNA transfection BBB cells. Interestingly. Paeonol is actively transported from the blood to brain across the BBB by a carrier mediated transporter system.

• Biomolecules & Therapeutics
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• 제27권1호
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• pp.107-116
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• 2019

The global obesity epidemic and associated metabolic diseases require alternative biological targets for new therapeutic strategies. In this study, we show that a phytochemical sulfuretin suppressed adipocyte differentiation of preadipocytes and administration of sulfuretin to high fat diet-fed obese mice prevented obesity and increased insulin sensitivity. These effects were associated with a suppressed expression of inflammatory markers, induced expression of adiponectin, and increased levels of phosphorylated ERK and AKT. To elucidate the molecular mechanism of sulfuretin in adipocytes, we performed microarray analysis and identified activating transcription factor 3 (Atf3) as a sulfuretin-responsive gene. Sulfuretin elevated Atf3 mRNA and protein levels in white adipose tissue and adipocytes. Consistently, deficiency of Atf3 promoted lipid accumulation and the expression of adipocyte markers. Sulfuretin's but not resveratrol's anti-adipogenic effects were diminished in Atf3 deficient cells, indicating that Atf3 is an essential factor in the effects of sulfuretin. These results highlight the usefulness of sulfuretin as a new anti-obesity intervention for the prevention of obesity and its associated metabolic diseases.

• Biomolecules & Therapeutics
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• 제29권6호
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• pp.615-629
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• 2021

An active compound, triterpene saponin, astersaponin I (AKNS-2) was isolated from Aster koraiensis Nakai (AKNS) and the autophagy activation and neuroprotective effect was investigated on in vitro and in vivo Parkinson's disease (PD) models. The autophagy-regulating effect of AKNS-2 was monitored by analyzing the expression of autophagy-related protein markers in SH-SY5Y cells using Western blot and fluorescent protein quenching assays. The neuroprotection of AKNS-2 was tested by using a 1-methyl-4-phenyl-2,3-dihydropyridium ion (MPP⁺)-induced in vitro PD model in SH-SY5Y cells and an MPTP-induced in vivo PD model in mice. The compound-treated SH-SY5Y cells not only showed enhanced microtubule-associated protein 1A/1B-light chain 3-II (LC3-II) and decreased sequestosome 1 (p62) expression but also showed increased phosphorylated extracellular signal-regulated kinases (p-Erk), phosphorylated AMP-activated protein kinase (p-AMPK) and phosphorylated unc-51-like kinase (p-ULK) and decreased phosphorylated mammalian target of rapamycin (p-mTOR) expression. AKNS-2-activated autophagy could be inhibited by the Erk inhibitor U0126 and by AMPK siRNA. In the MPP⁺-induced in vitro PD model, AKNS-2 reversed the reduced cell viability and tyrosine hydroxylase (TH) levels and reduced the induced α-synuclein level. In an MPTP-induced in vivo PD model, AKNS-2 improved mice behavioral performance, and it restored dopamine synthesis and TH and α-synuclein expression in mouse brain tissues. Consistently, AKNS-2 also modulated the expressions of autophagy related markers in mouse brain tissue. Thus, AKNS-2 upregulates autophagy by activating the Erk/mTOR and AMPK/mTOR pathways. AKNS-2 exerts its neuroprotective effect through autophagy activation and may serve as a potential candidate for PD therapy.

• Biomolecules & Therapeutics
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• 제29권2호
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• pp.195-204
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• 2021

Cereblon (CRBN), a substrate receptor of cullin 4-RING E3 ligase (CRL4) regulates the ubiquitination and degradation of c-Jun, mediating the lipopolysaccharide-induced cellular response. However, the upstream signaling pathway that regulates this process is unknown. In this study, we describe how endoplasmic reticulum (ER) stress reversely regulates sequestosome-1 (p62)and c-Jun protein levels. Furthermore, our study reveals that expression of p62 attenuates c-Jun protein levels through the ubiquitinproteasome system. Conversely, siRNA knockdown of p62 elevates c-Jun protein levels. Immunoprecipitation and immunoblotting experiments demonstrate that p62 interacts with c-Jun and CRBN to form a ternary protein complex. Moreover, we find that CRBN knockdown completely abolishes the inhibitory effect of p62 on c-Jun. Using brefeldin A as an inducer of ER stress, we demonstrate that the p62/c-Jun axis participates in the regulation of ER stress-induced apoptosis, and that CRBN is required for this regulation. In summary, we have identified an upstream signaling pathway, which regulates p62-mediated c-Jun degradation. Our findings elucidate the underlying molecular mechanism by which p62/c-Jun axis regulates the ER stress-induced apoptosis, and provide a new molecular connection between ER stress and apoptosis.

• Biomolecules & Therapeutics
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• 제30권3호
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• pp.238-245
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• 2022

Previous reports have demonstrated that genetic mechanisms greatly mediate responses to drugs of abuse, including methamphetamine (METH). The circadian gene Period 2 (Per2) has been previously associated with differential responses towards METH in mice. While the behavioral consequences of eliminating Per2 have been illustrated previously, Per2 overexpression has not yet been comprehensively described; although, Per2-overexpressing (Per2 OE) mice previously showed reduced sensitivity towards METH-induced addiction-like behaviors. To further elucidate this distinct behavior of Per2 OE mice to METH, we identified possible candidate biomarkers by determining striatal differentially expressed genes (DEGs) in both drug-naïve and METH-treated Per2 OE mice relative to wild-type (WT), through RNA sequencing. Of the several DEGs in drug naïve Per2 OE mice, we identified six genes that were altered after repeated METH treatment in WT mice, but not in Per2 OE mice. These results, validated by quantitative real-time polymerase chain reaction, could suggest that the identified DEGs might underlie the previously reported weaker METH-induced responses of Per2 OE mice compared to WT. Gene network analysis also revealed that Asic3, Hba-a1, and Rnf17 are possibly associated with Per2 through physical interactions and predicted correlations, and might potentially participate in addiction. Inhibiting the functional protein of Asic3 prior to METH administration resulted in the partial reduction of METH-induced conditioned place preference in WT mice, supporting a possible involvement of Asic3 in METH-induced reward. Although encouraging further investigations, our findings suggest that these DEGs, including Asic3, may play significant roles in the lower sensitivity of Per2 OE mice to METH.

• Biomolecules & Therapeutics
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• 제30권4호
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• pp.368-379
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• 2022

Hyaluronic acid (HA), a ligand of CD44, accumulates in some types of tumors and is responsible for tumor progression. The nuclear factor erythroid 2-like 2 (NRF2) regulates cytoprotective genes and drug transporters, which promotes therapy resistance in tumors. Previously, we showed that high levels of CD44 are associated with NRF2 activation in cancer stem like-cells. Herein, we demonstrate that HA production was increased in doxorubicin-resistant breast cancer MCF7 cells (MCF7-DR) via the upregulation of HA synthase-2 (HAS2). HA incubation increased NRF2, aldo-keto reductase 1C1 (AKR1C1), and multidrug resistance gene 1 (MDR1) levels. Silencing of HAS2 or CD44 suppressed NRF2 signaling in MCF7-DR, which was accompanied by increased doxorubicin sensitivity. The treatment with a HAS2 inhibitor, 4-methylumbelliferone (4-MU), decreased NRF2, AKR1C1, and MDR1 levels in MCF7-DR. Subsequently, 4-MU treatment inhibited sphere formation and doxorubicin resistance in MCF7-DR. The Cancer Genome Atlas (TCGA) data analysis across 32 types of tumors indicates the amplification of HAS2 gene is a common genetic alteration and is negatively correlated with the overall survival rate. In addition, high HAS2 mRNA levels are associated with increased NRF2 signaling and poor clinical outcome in breast cancer patients. Collectively, these indicate that HAS2 elevation contributes to chemoresistance and sphere formation capacity of drug-resistant MCF7 cells by activating CD44/NRF2 signaling, suggesting a potential benefit of HAS2 inhibition.

• Biomolecules & Therapeutics
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• 제30권4호
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• pp.348-359
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• 2022

Gastric adenocarcinoma is among the top causes of cancer-related death and is one of the most commonly diagnosed carcinomas worldwide. Benzyl isothiocyanate (BITC) has been reported to inhibit the gastric cancer metastasis. In our previous study, BITC induced apoptosis in AGS cells. The purpose of the present study was to investigate the effect of BITC on autophagy mechanism in AGS cells. First, the AGS cells were treated with 5, 10, or 15 μM BITC for 24 h, followed by an analysis of the autophagy mechanism. The expression level of autophagy proteins involved in different steps of autophagy, such as LC3B, p62/SQSTM1, Atg5-Atg12, Beclin1, p-mTOR/mTOR ratio, and class III PI3K was measured in the BITC-treated cells. Lysosomal function was investigated using cathepsin activity and Bafilomycin A1, an autophagy degradation stage inhibitor. Methods including qPCR, western blotting, and immunocytochemistry were employed to detect the protein expression levels. Acridine orange staining and omnicathepsin assay were conducted to analyze the lysosomal function. siRNA transfection was performed to knock down the LC3B gene. BITC reduced the level of autophagy protein such as Beclin 1, class III PI3K, and Atg5-Atg12. BITC also induced lysosomal dysfunction which was shown as reducing cathepsin activity, protein level of cathepsin, and enlargement of acidic vesicle. Overall, the results showed that the BITC-induced AGS cell death mechanism also comprises the inhibition of the cytoprotective autophagy at both initiation and degradation steps.