• BMB Reports
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• 제54권12호
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• pp.608-613
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• 2021

Melanoma, the most serious type of skin cancer, exhibits a high risk of metastasis. Although chemotherapeutic treatment for metastatic melanoma improves disease outcome and patient survival, some patients exhibit resistance or toxicity to the drug treatment regime. OTUB1 is a deubiquitinating enzyme overexpressed in several cancers. In this study, we investigated the effects of inhibiting OTUB1 expression on melanoma-cell proliferation and viability and identified the underlying molecular mechanism of action of OTUB1. We did endogenous OTUB1 knockdown in melanoma cells using short interfering RNA, and assessed the resulting phenotypes via MTT assays, Western blotting, and cell-cycle analysis. We identified differentially expressed genes between OTUB1-knockdown cells and control cells using RNA sequencing and confirmed them via Western blotting and reverse transcription polymerase chain reaction. Furthermore, we investigated the involvement of apoptotic and cell survival signaling pathways upon OTUB1 depletion. OTUB1 depletion in melanoma cells decreased cell viability and caused simultaneous accumulation of cells in the sub-G1 phase, indicating an increase in the apoptotic-cell population. RNA sequencing of OTUB1-knockdown cells revealed an increase in the levels of the apoptosis-inducing protein TRAIL. Additionally, OTUB1-knockdown cells exhibited increased sensitivity to PLX4032, a BRAF inhibitor, implying that OTUB1 and BRAF act collectively in regulating apoptosis. Taken together, our findings show that OTUB1 induces apoptosis of melanoma cells in vitro, likely by upregulating TRAIL, and suggest that approaches targeting OTUB1 can be developed to provide novel therapeutic strategies for treating melanoma.

• Journal of Ginseng Research
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• 제48권1호
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• pp.52-58
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• 2024

Background: Skeletal muscle denervation leads to motor neuron degeneration, which in turn reduces muscle fiber volumes. Recent studies have revealed that apoptosis plays a role in regulating denervation-associated pathologic muscle wasting. Korean red ginseng (KRG) has various biological activities and is currently widely consumed as a medicinal product worldwide. Among them, ginseng has protective effects against muscle atrophy in in vivo and in vitro. However, the effects of KRG on denervation-induced muscle damage have not been fully elucidated. Methods: We induced skeletal muscle atrophy in mice by dissecting the sciatic nerves, administered KRG, and then analyzed the muscles. KRG was administered to the mice once daily for 3 weeks at 100 and 400 mg/kg/day doses after operation. Results: KRG treatment significantly increased skeletal muscle weight and tibialis anterior (TA) muscle fiber volume in injured areas and reduced histological alterations in TA muscle. In addition, KRG treatment reduced denervation-induced apoptotic changes in TA muscle. KRG attenuated p53/Bax/cytochrome c/Caspase 3 signaling induced by nerve injury in a dose-dependent manner. Also, KRG decreases protein kinase B/mammalian target of rapamycin pathway, reducing restorative myogenesis. Conclusion: Thus, KRG has potential protective role against denervation-induced muscle atrophy. The effect of KRG treatment was accompanied by reduced levels of mitochondria-associated apoptosis.

• Parasites, Hosts and Diseases
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• 제62권1호
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• pp.30-41
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• 2024

The dense granule protein of Toxoplasma gondii, inhibitor of signal transducer and activator of transcription 1 (IST) is an inhibitor of signal transducer and activator of transcription 1 (STAT1) transcriptional activity that binds to STAT1 and regulates the expression of inflammatory molecules in host cells. A sterile inflammatory liver injury in pathological acute liver failures occurs when excessive innate immune function, such as the massive release of IFN-γ and TNF-α, is activated without infection. In relation to inflammatory liver injury, we hypothesized that Toxoplasma gondii inhibitor of STAT1 transcription (TgIST) can inhibit the inflammatory response induced by activating the STAT1/IRF-1 mechanism in liver inflammation. This study used IFN-γ and TNF-α as inflammatory inducers at the cellular level of murine hepatocytes (Hepa-1c1c7) to determine whether TgIST inhibits the STAT1/IRF-1 axis. In stable cells transfected with TgIST, STAT1 expression decreased with a decrease in interferon regulatory factor (IRF)-1 levels. Furthermore, STAT1 inhibition of TgIST resulted in lower levels of NF-κB and COX2, as well as significantly lower levels of class II transactivator (CIITA), iNOS, and chemokines (CLXCL9/10/11). TgIST also significantly reduced the expression of hepatocyte proapoptotic markers (Caspase3/8/9, P53, and BAX), which are linked to sterile inflammatory liver injury. TgIST also reduced the expression of adhesion (ICAM-1 and VCAM-1) and infiltration markers of programmed death-ligand 1 (PD-L1) induced by hepatocyte and tissue damage. TgIST restored the cell apoptosis induced by IFN-γ/TNF-α stimulation. These results suggest that TgIST can inhibit STAT1-mediated inflammatory and apoptotic responses in hepatocytes stimulated with proinflammatory cytokines.

• 한국식품영양과학회지
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• 제32권3호
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• pp.437-443
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• 2003

선행연구에서 라이코펜이 HT-29세포의 증식을 억제하는 것을 관찰하였기 때문에 본 연구는 그 기전을 연구하기 위하여 수행되 었다. 라이코펜이 HT-29 세포의 사멸을 유도하는지 조사하기 위해서 여러 농도의 라이코펜이 포함된 배지에서 세포를 4일 동안 배양하였다. 라이코펜 농도의 증가에 따라 사멸되는 세포에서 나타나는 특징의 하나인 DNA fragmentation이 증가하는 것을 관찰하였다. Western blot을 수행하여 얻은 결과에 의하면 라이코펜이 IGF-IR, IRS-1, PI3K, Akt와 같은 IGF-IR pathway에 속하는 단백질의 수준을 감소시켰다. IGF-IR의 인산화를 유도하기 위해서 라이코펜이 포함된 배지에서 세포를 배양하고 IGF-I을 첨가하여 0, 5, 10, 60분간 배양한 다음 IGF-IR antibody를 이용하여 immunoprecipitation을 수행하였다. 라이코펜은 IGF-I에 의한 IGF-IR, IRS-1의 인산화와 IGF-IR와 PI3K의 결합을 감소하고 인산화된 Akt를 감소시켰다. 이와 같은 IGF-IR signaling의 억제는 이 대장암세포에 존재하는 IGF-II의 autocrine loop을 억제하는 원인이 될 수 있어, 라이코펜의 암세포증식을 억제하는 기전 중의 하나가 될 수 있다. 라이코펜은 토마토와 그 가공품에 많이 존재하는 물질로 자연적인 식사를 통해 많이 섭취할 수 있는 물질이다. 라이코펜의 항암 기전을 밝혀냄으로써 미래 암예방과 치료를 위한 중요한 기능성 영양소를 생산할 수 있는 기초를 마련해줄 수 있을 것으로 기대된다.

• Biomolecules & Therapeutics
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• 제23권1호
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• pp.31-38
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• 2015

Histone acetylation plays a critical role in the regulation of transcription by altering the structure of chromatin, and it may influence the resistance of some tumor cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by regulating the gene expression of components of the TRAIL signaling pathway. In this study, we investigated the effects and molecular mechanisms of trichostatin A (TSA), a histone deacetylase inhibitor, in sensitizing TRAIL-induced apoptosis in Caki human renal carcinoma cells. Our results indicate that nontoxic concentrations of TSA substantially enhance TRAIL-induced apoptosis compared with treatment with either agent alone. Cotreatment with TSA and TRAIL effectively induced cleavage of Bid and loss of mitochondrial membrane potential (MMP), which was associated with the activation of caspases (-3, -8, and -9) and degradation of poly (ADP-ribose) polymerase (PARP), contributing toward the sensitization to TRAIL. Combined treatment with TSA and TRAIL significantly reduced the levels of the cellular Fas-associated death domain (FADD)-like interleukin-$1{\beta}$-converting enzyme (FLICE) inhibitory protein (c-FLIP), whereas those of death receptor (DR) 4, DR5, and FADD remained unchanged. The synergistic effect of TAS and TRAIL was perfectly attenuated in c-$FLIP_L$-overexpressing Caki cells. Taken together, the present study demonstrates that down-regulation of c-FLIP contributes to TSA-facilitated TRAIL-induced apoptosis, amplifying the death receptor, as well as mitochondria-mediated apoptotic signaling pathways.

• Asian Pacific Journal of Cancer Prevention
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• 제15권11호
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• pp.4519-4525
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• 2014

Oleanolic acid (OA) is a nutritional component widely distributed in various vegetables. Although it has been well recognized for decades that OA exerts certain anti-tumor activity by inducing mitochondria-dependent apoptosis, it is still unclear that what molecular signaling is responsible for this effect. In this study, we employed cancer cell lines, A549, BXPC-3, PANC-1 and U2OS to elucidate the molecular mechanisms underlying OA anti-tumor activity. We found that activation of MAPK pathways, including p-38 MAPK, JNK and ERK, was triggered by OA in both a dose and time-dependent fashion in all the tested cancer cells. Activation was accompanied by cleavage of caspases and PARP as well as cytochrome C release. SB203580 (p38 MAPK inhibitor), but not SP600125 (JNK inhibitor) and U0126 (ERK inhibitor), rescued the pro-apoptotic effect of OA on A549 and BXPC-3 cells. OA induced p38 MAPK activation promoted mitochondrial translocation of Bax and Bim, and inhibited Bcl-2 function by enhancing their phosphorylation. OA can induce reactive oxygen species (ROS)-dependent ASK1 activation, and this event was indispensable for p38 MAPK-dependent apoptosis in cancer cells. In vivo, p38 MAPK knockdown A549 tumors proved resistant to the growth-inhibitory effect of OA. Collectively, we elucidated that activation of ROS/ASK1/p38 MAPK pathways is responsible for the apoptosis stimulated by OA in cancer cells. Our finding can contribute to a better understanding of molecular mechanisms underlying the antitumor activity of nutritional components.

• Molecules and Cells
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• 제43권5호
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• pp.459-468
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• 2020

Nuclear receptor subfamily group H member 4 (NR1H4), also known as farnesoid X receptor, has been implicated in several cellular processes in the liver and intestine. Preclinical and clinical studies have suggested a role of NR1H4 in colon cancer development; however, how NR1H4 regulates colon cancer cell growth and survival remains unclear. We generated NR1H4 knockout (KO) colon cancer cells using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (CAS9) technology and explored the effects of NR1H4 KO in colon cancer cell proliferation, survival, and apoptosis. Interestingly, NR1H4 KO cells showed impaired cell proliferation, reduced colony formation, and increased apoptotic cell death compared to control colon cancer cells. We identified MYC as an important mediator of the signaling pathway alterations induced by NR1H4 KO. NR1H4 silencing in colon cancer cells resulted in reduced MYC protein levels, while NR1H4 activation using an NR1H4 ligand, chenodeoxycholic acid, resulted in time- and dose-dependent MYC induction. Moreover, NR1H4 KO enhanced the anti-cancer effects of doxorubicin and cisplatin, supporting the role of MYC in the enhanced apoptosis observed in NR1H4 KO cells. Taken together, our findings suggest that modulating NR1H4 activity in colon cancer cells might be a promising alternative approach to treat cancer using MYC-targeting agents.

• Biomolecules & Therapeutics
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• 제25권5호
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• pp.504-510
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• 2017

Inhibitor of nuclear factor kappa-B kinase beta ($IKK{\beta}$) plays a critical role in cell proliferation and inflammation in various cells by activating $NF-{\kappa}B$ signaling. However, the interrelationship between peroxisome proliferator-activated receptor ${\alpha}$ ($PPAR{\alpha}$) and $IKK{\beta}$ in cell proliferation is not clear. In this study, we investigated the possible role of $PPAR{\alpha}$ in the hepatic cell death in the absence of $IKK{\beta}$ gene using liver-specific Ikkb-null ($Ikkb^{F/F-AlbCre}$) mice. To examine the function of $PPAR{\alpha}$ activation in hepatic cell death, wild-type ($Ikkb^{F/F}$) and $Ikkb^{F/F-AlbCre}$ mice were treated with $PPAR{\alpha}$ agonist Wy-14,643 (0.1% w/w chow diet) for two weeks. As a result of Wy-14,643 treatment, apoptotic markers including caspase-3 cleavage, poly (ADP-ribose) polymerase (PARP) cleavage and TUNEL-positive staining were significantly decreased in the $Ikkb^{F/F-AlbCre}$ mice. Surprisingly, Wy-14,643 increased the phosphorylation of p65 and STAT3 in both Ikkb and $Ikkb^{F/F-AlbCre}$ mice. Furthermore, BrdU-positive cells were significantly increased in both groups after treatment with Wy-14,643. Our results suggested that $IKK{\beta}-derived$ hepatic apoptosis could be altered by $PPAR{\alpha}$ activation in conjunction with activation of $NF-{\kappa}B$ and STAT3 signaling.

• Asian Pacific Journal of Cancer Prevention
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• 제14권12호
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• pp.7501-7508
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• 2013

Background: Chronic myeloid leukemia (CML) is a myeloproliferative disorder of hematopoietic stem cell scarrying the Philadelphia (Ph) chromosome and an oncogenic BCR-ABL1 fusion gene. The tyrosine kinase inhibitor (TKI) of BCR-ABL1 kinase is a treatment of choice for control of CML. Objective: Recent studies have demonstrated that miRNAs within exosomes from cancer cells play crucial roles in initiation and progression. This study was performed to assess miRNAs within exosomes of K562 cells. Methods: miRNA microarray analysis of K562 cells and K562 cell-derived exosomes was conducted with the 6th generation miRCURYTM LNA Array (v.16.0). Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were also carried out. GO terms and signaling pathways were categorized into 66 classes (including homophilic cell adhesion, negative regulation of apoptotic process, cell adhesion) and 26 signaling pathways (such as Wnt). Results: In exosomes, 49 miRNAs were up regulated as compared to K562 cells, and two of them were further confirmed by quantitative real-time PCR. There are differentially expressed miRNAs between K562 cell derived-exosomes and K562 cells. Conclusion: Selectively expressed miRNAs in exosomes may promote the development of CML via effects on interactions (e.g. adhesion) of CML cells with their microenvironment.

• Journal of Microbiology and Biotechnology
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• 제29권11호
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• pp.1830-1840
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• 2019

Loliolide is one of the most ubiquitous monoterpenoid compounds found in algae, and its potential therapeutic effect on various dermatological conditions via agent-induced biological functions, including anti-oxidative and anti-apoptotic properties, was demonstrated. Here, we investigated the effects of loliolide on hair growth in dermal papilla (DP) cells, the main components regulating hair growth and loss conditions. For this purpose, we used a three-dimensional (3D) DP spheroid model that mimics the in vivo hair follicle system. Biochemical assays showed that low doses of loliolide increased the viability and size of 3D DP spheroids in a dose-dependent manner. This result correlated with increases in expression levels of hair growth-related autocrine factors including VEGF, IGF-1, and KGF. Immunoblotting and luciferase-reporter assays further revealed that loliolide induced AKT phosphorylation, and this effect led to stabilization of β-catenin, which plays a crucial role in the hair-inductive properties of DP cells. Further experiments showed that loliolide increased the expression levels of the DP signature genes, ALP, BMP2, VCAN, and HEY1. Furthermore, conditioned media from loliolide-treated DP spheroids significantly enhanced proliferation and the expression of hair growth regulatory genes in keratinocytes. These results suggested that loliolide could function in the hair growth inductivity of DP cells via the AKT/β-catenin signaling pathway.