• Journal of Microbiology and Biotechnology
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• 제18권12호
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• pp.1997-2003
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• 2008

Cordyceps militaris is well known as a traditional medicinal mushroom and has been shown to exhibit immunostimulatory and anticancer activities. In this study, we investigated the apoptosis induced by an aqueous extract of C. militaris (AECM) via the activation of caspases and altered mitochondrial membrane permeability in human breast cancer MDA-MB-231 cells. Exposure to AECM induced apoptosis, as demonstrated by a quantitative analysis of nuclear morphological change and a flow cytometric analysis. AECM increased hyperpolarization of mitochondrial membrane potential and promoted the activation of caspases. Both the cytotoxic effect and apoptotic characteristics induced by AECM treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role of caspase-3 in the observed cytotoxic effect. AECM-induced apoptosis was associated with the inhibition of Akt activation in a time-dependent manner, and pretreatment with LY294002, a PI3K/Akt inhibitor, significantly increased AECM-induced apoptosis. The results indicated that AECM-induced apoptosis may relate to the activation of caspase-3 and mitochondria dysfunctions that correlate with the inactivation of Akt.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 2002년도 창립10주년기념 및 국립독성연구원 의약품동등성평가부서 신설기념 국재학술대회:생물학적 동등성과 의약품 개발 전략을 위한 국제심포지움
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• pp.238-238
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• 2002

We studied whether kinase pathways are involved in TCDD-induced gene expression by treating specific kinase inhibitors ncluding MEK1 inhibitor PD98059, p38 inhibitor SB202190, PI-3 kinase inhibitor Wortmannin or LY294002 or protein tyrosine kinase inhibitor Genestein and then tested the effects of individual inhibitors on TCDD-induced gene expression of cytochromelAl gene (CYPlAl). Our results show that PD98059, MEK-1 inhibitor reduces dioxin-inducible transcription of CYPlAl. p44/p42MAPK, that is phosphorylated by Mek-1, are phosphorlylated by treatment of TCDD, peaking at lnM, 30min treatments. Overexpressions of p44/p42 MAPK dominant negative mutants suppress dioxin dependent transcription of DRE-driven reporter gene in a dose-dependent manner. Our results demonstrate that p44/p42 MAPK is essential for transcriptional activity of AHR/ARNT heterodimer. We found that PD98059 dose-dependently blocks TCDD-induced DRE binding of the AHR/ARNT heterodimer, thereby it reduces TCDD-induced gene expression. Therefore, our results indicate that Mek-1/p44/p42 MAPK pathway is involved in TCDD-induced gene expression, [This study was supported by a grant from Korean Research Foundation Grant (X01529)to H. Park]

• Molecules and Cells
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• 제23권2호
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• pp.198-206
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• 2007

Hydroquinone is a toxic compound and a major benzene metabolite. We report that it strongly inhibits the activation of macrophages and associated cells. Thus, it suppressed the production of proinflammatory cytokines [tumor necrosis factor (TNF)-${\alpha}$, interleukin (IL)-$1{\beta}$, IL-3, IL-6, IL-10, IL-12p40, IL-23], secretion of toxic molecules [nitric oxide (NO) and reactive oxygen species (ROS)] and the activation and expression of CD29 as judged by cell-cell adhesion and surface staining experiments. The inhibition was due to the induction of heme oxygenase (HO)-1 in LPS-activated macrophages, since blocking HO-1 activity with ZnPP, an HO-1 specific inhibitor, abolished hydroquinone's NO inhibitory activity. In addition, hydroquinone and inhibitors (wortmannin and LY294002) of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway had very similar inhibitory effects on LPS-induced and CD29-mediated macrophage responses, including the phoshorylation of Akt. Therefore, our data suggest that hydroquinone inhibits macrophage-mediated immune responses by modulating intracellular signaling and protective mechanisms.

• Molecules and Cells
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• 제26권1호
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• pp.48-52
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• 2008

We here demonstrate an anti-inflammatory action of raloxifene, a selective estrogen receptor modulator, in lipopolysaccharide (LPS)-induced murine macrophage RAW264.7 cells. Treatment with raloxifene at micromolar concentrations suppressed the production of nitric oxide (NO) by down-regulating expression of the inducible nitric oxide synthase (iNOS) gene in LPS-activated cells. The decreased expression of iNOS and subsequent reduction of NO were due to inhibition of nuclear translocation of transcription factor NF-${\kappa}B$. These effects were significantly inhibited by exposure to the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, or by expression of a dominant negative mutant of PI 3-kinase. In addition, pretreatment with raloxifene reduced LPS-induced Akt phosphorylation as well as NF-${\kappa}B$ DNA binding activity and NF-${\kappa}B$-dependent reporter gene activity. Thus our findings indicate that raloxifene exerts its anti-inflammatory action in LPS-stimulated macrophages by blocking the PI 3-kinase-Akt-NF-${\kappa}B$ signaling cascade, and eventually reduces expression of pro-inflammatory genes such as iNOS.

• 대한의생명과학회지
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• 제15권1호
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• pp.61-66
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• 2009

Leflunomide is an immunomodulatory agent used for the treatment of rheumatoid arthritis (RA). Leflunomide known as a regulator of iNOS synthesis which largely decreases NO production in diverse cell type. However, the effect of leflunomide on chondrocyte is still poorly understood. In our previous studies, we have shown that direct production of Nitric oxide (NO) by treating chondrocytes with NO donor, sodium nitroprusside (SNP), causes apoptosis via p38 mitogen-activated protein kinase in association with elevation of p53 protein level, caspase-3 activation. In this study, we characterized the molecular mechanism by which A77 1726 inhibit apoptosis. We found that A77 1726 inhibit NO-induced apoptosis as determined by MTT (Thiazolyl Blue Tetrazolium Bromide) assay and DNA fragmentation. The inhibition of apoptosis by A77 1726 was accompanied by increased PI-3 kinase and AKT activities. So, inhibition of phosphatidylinositol (PI)-3kinase with LY294002 rescued apoptosis. Triciribine, the specific inhibitor of AKT, also abolished anti-apoptotic effect. Our results indicate that A77 1726, the active metabolite of leflunomide, mediates NO-induced apoptosis in chondrocytes by modulating up-regulation of PI-3 kinase and AKT.

• Molecules and Cells
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• 제21권2호
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• pp.294-301
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• 2006

We have previously reported that phosphorylation of eukaryotic elongation factor 2 (eEF2) is related to the differentiation of chick embryonic muscle cells in culture. In the present study, we found that eEF2 phosphorylation declined shortly after induction of differentiation of L6 myoblasts, when the cells prepare for terminal differentiation by withdrawing from the cell cycle. This decrease in phosphorylation was prevented by inhibitors of phosphoinositide 3-kinase (PI3-kinase) that strongly inhibit myoblast differentiation. We hypothesized that PI3-kinase plays an important role in myoblast differentiation by regulating eEF2 phosphorylation in the early stages of differentiation. To test this hypothesis, myoblasts were synchronized at in $G_2/M$ and cultured in fresh differentiation medium (DM) or growth medium (GM). In DM the released cells accumulated in $G_0$/$G_1$ while in GM they progressed to S phase. In addition, cyclin D1 was more rapidly degraded in DM than in GM, and eEF2 phosphorylation decreased more. Inhibitors of PI3-kinase increased eEF2 phosphorylation, but PI3-kinase became more activated when eEF2 phosphorylation declined. These results suggest that the regulation of L6 myoblast differentiation by PI3-kinase is related to eEF2 phosphorylation.

• Biomolecules & Therapeutics
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• 제22권6호
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• pp.497-502
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• 2014

In the present study, we found that the natural compound arctigenin inhibited hydrogen peroxide-induced reactive oxygen species (ROS) production in rat primary astrocytes. Since hemeoxygenase-1 (HO-1) plays a critical role as an antioxidant defense factor in the brain, we examined the effect of arctigenin on HO-1 expression in rat primary astrocytes. We found that arctigenin increased HO-1 mRNA and protein levels. Arctigenin also increases the nuclear translocation and DNA binding of Nrf2/c-Jun to the antioxidant response element (ARE) on HO-1 promoter. In addition, arctigenin increased ARE-mediated transcriptional activities in rat primary astrocytes. Further mechanistic studies revealed that arctigenin increased the phosphorylation of AKT, a downstream substrate of phosphatidylinositol 3-kinase (PI3K). Treatment of cells with a PI3K-specific inhibitor, LY294002, suppressed the HO-1 expression, Nrf2 DNA binding and ARE-mediated transcriptional activities in arctigenin-treated astrocyte cells. The results collectively suggest that PI3K/AKT signaling pathway is at least partly involved in HO-1 expression by arctigenin via modulation of Nrf2/ARE axis in rat primary astrocytes.

• 대한의생명과학회지
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• 제27권3호
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• pp.170-176
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• 2021

Thapsigargin (TG), a sarco/endoplasmic reticulum (ER) Ca²⁺-ATPase (SERCA) inhibitor, has been widely used as an agonist for platelet aggregation for decades. In this study, we investigated the effect of TG on the release of lactate dehydrogenase (LDH) for platelets and elucidated its mechanism. Platelet LDH release and platelet aggregation were increased by TG treatment; 1,000 nM of TG induced the complete lysis of platelets. Other agonists such as collagen (2.5 ㎍/mL), thrombin (0.1 U/mL), and ADP (10 mM) did not induce significant platelet LDH release despite platelet aggregation. Finally, we investigated the effects of pharmacological inhibitors on TG-induced platelet aggregation and LDH release. SP600125, a JNK inhibitor, and LY294002, a PI-3K inhibitor, inhibited TG-induced platelet LDH release but not platelet aggregation. Forskolin, an adenylyl cyclase activator, also inhibited LDH release without affecting platelet aggregation by TG. These results suggest that the TG-induced platelet aggregation was accompanied by LDH release but regulated by a different signaling pathway.

• Biomolecules & Therapeutics
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• 제27권5호
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• pp.484-491
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• 2019

Glioblastoma is the most aggressive common brain tumor in adults. Curcumin, from Curcuma longa, is an effective antitumor agent. Although the same proteins control both autophagy and cell death, the molecular connections between them are complicated and autophagy may promote or inhibit cell death. We investigated whether curcumin affects autophagy, which regulates curcumin-mediated tumor cell death in A172 human glioblastoma cells. When A172 cells were incubated with $10{\mu}M$ curcumin, autophagy increased in a time-dependent manner. Curcumin-induced cell death was reduced by co-incubation with the autophagy inhibitors 3-methyladenine (3-MA), hydroxychloroquine (HCQ), and LY294002. Curcumin-induced cell death was also inhibited by co-incubation with rapamycin, an autophagy inducer. When cells were incubated under serum-deprived medium, LC3-II amount was increased but the basal level of cell viability was reduced, leading to the inhibition of curcumin-induced cell death. Cell death was decreased by inhibiting curcumin-induced autophagy using small interference RNA (siRNA) of Atg5 or Beclin1. Therefore, curcumin-mediated tumor cell death is promoted by curcumin-induced autophagy, but not by an increase in the basal level of autophagy in rapamycin-treated or serum-deprived conditions. This suggests that the antitumor effects of curcumin are influenced differently by curcumin-induced autophagy and the prerequisite basal level of autophagy in cancer cells.

• 한국식품영양과학회지
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• 제45권11호
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• pp.1589-1594
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• 2016

본 연구는 후추과 식물의 주성분인 piperine이 위암세포 AGS의 항암 효과에 미치는 영향을 확인하기 위해 수행되었다. Piperine에 의한 위암세포 AGS의 생존율을 확인하기 위해 MTT assay를 수행한 결과 농도 의존적으로 암세포의 생존율이 감소하는 것을 확인하였다. 이러한 암세포의 생존율 억제가 apoptosis에 의한 효과인지를 확인하기 위해 DAPI staining을 실시한 결과 piperine을 처치한 군에서 apoptotic body와 염색질 응축이 증가하는 것을 확인하였다. MTT assay와 DAPI staining의 결과를 바탕으로 piperine이 위암세포 AGS에서 apoptosis와 관련한 단백질 발현 양상에 미치는 영향을 확인하기 위해 western blotting을 실시하였다. 그 결과 piperine에 의해 AGS 세포에서 apoptosis를 유도하는 p53, Bax의 발현은 농도 의존적으로 증가하였고, apoptosis를 억제하는 Bcl-2, XIAP의 발현은 농도 의존적으로 감소하였다. Apoptosis의 궁극적 단계인 caspase-9와 손상된 DNA를 복구하는 PARP의 분절은 증가하였으며, apoptosis를 방해하는 인자인 Akt의 인산화는 농도 의존적으로 감소하였다. Akt 억제제인 LY294002와 piperine을 병행 또는 단독으로 처치하여 western blotting을 진행한 결과 Bcl-2의 발현은 piperine을 단독으로 처치했을 때와 유사한 감소 경향을 보였지만, p-Akt의 발현은 대조군과 비교해 LY294002와 piperine을 단독으로 처치했을 때보다 병행했을 때 더욱 감소하였다. 이와 반대로 Bax와 cleaved-PARP의 발현은 강하게 증가하였다. 본 연구의 결과를 종합해볼 때 piperine이 위암세포 AGS에서 Akt 경로를 통해 apoptosis를 유도하는 것으로 여겨지며, 향후 위암 예방제나 치료제로의 개발 가능성이 있을 것으로 생각한다.