• IMMUNE NETWORK
• /
• v.1 no.3
• /
• pp.196-202
• /
• 2001

Backgorund: WEHI-231 B cell line is a representative model for $IgM^+$ mature B cells. To understand the signaling differences between mature and immature B cells, we compared the responsiveness of WEHI-231 and Bal 17 B cell lines to BCR cross-linking. Methods: The extents of tyrosine phosphorylation, ligand-induced internalization, and activation-induced cell death upon BCR cross-linking were compared in two cell lines. Results: Despite a higher expression of BCR, cross-linking of BCR on WEHI-231 cell evoked a weaker level of tyrosine phosphorylation and BCR endocytosis than Bal 17 cells. Furthermore, the endocytosed BCR could not enter the lysosomal compartment and stayed as peripheral spots in WEHI-231 cells. Conclusion: WEHI-231 cell showed preferred BCR-mediated signaling pathways leading to a reduced capability of antigen presentation as well as the enhanced apoptosis in comparision with Bal 17 cells. These results might reflect the signaling differences between mature and immature B cells.

• The Journal of Internal Korean Medicine
• /
• v.32 no.4
• /
• pp.565-583
• /
• 2011

Objectives : This study investigated the biochemical mechanisms of anti-proliferative and pro-apoptotic effects of the water extract of Dan-Seon-Tang (DST) in human leukemia U937 cells. Methods : U937 cells were exposed to DST and growth inhibition was measured by MTT assay. Results : Exposure of U937 cells to DST resulted in the growth inhibition in a concentration-dependent manner. This inhibitory effect was associated with morphological changes and apoptotic cell death such as formation of apoptotic bodies, increased populations of apoptotic-sub G1 phase and induction of DNA fragmentation. The induction of apoptotic cell death in U937 cells by DST was associated with up-regulation of death receptor 4 (DR4) and down-regulation of Bid, surviving and cellular inhibition of apoptosis protein-2 (cIAP-2) expression. DST treatment also induced the proteolytic activation of caspase-3, caspase-8 and caspase-9, and a concomitant degradation of caspase-3 substrate proteins such as poly (ADP-ribose) polymerase (PARP), phospholipase (PLC)-${\gamma}1$, ${\beta}$-catenin and DNA fragmentation factor 45/inhibotor of caspase activated DNAse (DFF45/ICAD). Furthermore, apoptotic cell death by DST was significantly inhibited by caspase-3 specific inhibitor z-DEVD-fmk, demonstrating the important role of caspase-3. Conclusions : These findings suggest that herb prescription DST may be a potential chemotherapeutic agent for the control of human leukemia U937 cells; further study is needed to identify the active compounds.

• Biomolecules & Therapeutics
• /
• v.22 no.3
• /
• pp.184-192
• /
• 2014

${\beta}$-lapachone is a naturally occurring quinone that selectively induces apoptotic cell death in a variety of human cancer cells in vitro and in vivo; however, its mechanism of action needs to be further elaborated. In this study, we investigated the effects of ${\beta}$-lapachone on the induction of apoptosis in human gastric carcinoma AGS cells. ${\beta}$-lapachone significantly inhibited cellular proliferation, and some typical apoptotic characteristics such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells were observed in ${\beta}$-lapachone-treated AGS cells. Treatment with ${\beta}$-lapachone caused mitochondrial transmembrane potential dissipation, stimulated the mitochondria-mediated intrinsic apoptotic pathway, as indicated by caspase-9 activation, cytochrome c release, Bcl-2 downregulation and Bax upregulation, as well as death receptor-mediated extrinsic apoptotic pathway, as indicated by activation of caspase-8 and truncation of Bid. This process was accompanied by activation of caspase-3 and concomitant with cleavage of poly(ADP-ribose) polymerase. The general caspase inhibitor, z-VAD-fmk, significantly abolished ${\beta}$-lapachone-induced cell death and inhibited growth. Further analysis demonstrated that the induction of apoptosis by ${\beta}$-lapachone was accompanied by inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The PI3K inhibitor LY29004 significantly increased ${\beta}$-lapachone-induced apoptosis and growth inhibition. Taken together, these findings indicate that the apoptotic activity of ${\beta}$-lapachone is probably regulated by a caspase-dependent cascade through activation of both intrinsic and extrinsic signaling pathways, and that inhibition of the PI3K/Akt signaling may contribute to ${\beta}$-lapachone-mediated AGS cell growth inhibition and apoptosis induction.

• Proceedings of the Plant Resources Society of Korea Conference
• /
• 2020.08a
• /
• pp.77-77
• /
• 2020

Purple loosestrife-Lythrum anceps (Koehne) Makino is a herbaceous perennial plant belonging to the Lythraceae family. It has been used for centuries in Korea and other Asian traditional medicine. It has been showed pharmacological effects, including anti-oxidant and anti-microbial effects. However, the mechanisms underlying its anti-cancer mechanisms are not yet understood. In this study, we investigated the mechanism of apoptosis signaling pathways by ethanol extract of Lythrum anceps (Koehne) Makino (ELM) in human leukemia U937 cells. Treatment with ELM significantly inhibited cell growth in a dose-dependent manner by inducing apoptosis, as evidenced by the formation of apoptotic bodies (ApoBDs), DNA fragmentation and increased populations of sub-G1 ratio. Induction of apoptosis by ELM was connected with up-regulation of death receptor (DR) 4 and DR5, pro-apoptotic Bax protein expression and down-regulation of anti-apoptotic Bcl-2 protein, and inhibitor of apoptosis protein (IAP) family proteins (XIAP, cIAP-1, survivin), depending on dosage. This induction was associated with Bid truncation, mitochondrial dysfunction, proteolytic activation of caspases (-3, -8 and -9) and cleavage of poly(ADP-ribose) polymerase protein. Therefore, our data indicate that ELM suppresses U937 cell growth by activating the intrinsic and extrinsic apoptosis pathways, and thus may have applications as a potential source for an anti-leukemic chemotherapeutic agent.

• Journal of Life Science
• /
• v.26 no.7
• /
• pp.868-874
• /
• 2016

Receptor-interacting protein kinase 1 (RIPK1) and RIPK3 are members of the serine or threonine protein kinase superfamily that phosphorylates the hydroxyl group of serine or threonine through the highly conserved kinase region. The RIPK family plays a crucial role not only in inflammation and innate immunity, but also in mediating programmed cell death, such as apoptosis and necroptosis. The interaction between RIPK1 and other TNFR1-related proteins has been shown to assemble a signaling complex I that controls activation of the pro-survival transcription factor NF-κB upon binding of cytokines to TNF receptor 1 (TNFR1). Moreover, RIPK1 and RIPK3 interact through their RIP homotypic interaction motifs (RHIMs) to mediate programmed necrosis, which has long been considered an accidental and uncontrolled cell death form with morphological characteristics differing from those of apoptosis. Highly conserved sequences of RHIM in RIPK1 and RIPK3 were shown to regulate their binary interaction, leading to assembly of a cytosolic amyloid complex termed the “necrosome”. The necrosome also contains mixed lineage kinase domain-like protein (MLKL), which has been found recently to be a substrate of RIPK3 to mediate downstream signaling. This review provides an overview of the functional and physiological characteristics of RIPKs and MLKL in TNF signaling.

• International Journal of Oral Biology
• /
• v.45 no.3
• /
• pp.107-114
• /
• 2020

Acacetin, which is present in damiana (Turnera diffusa) and black locust (Robinia pseudoacacia), has several pharmacologic activities such as antioxidant, anti-inflammatory, and anti-proliferative effects on cancer cells. However, the effect of acacetin on head and neck cancers has not been clearly established. This study aimed to examine the effects of acacetin on cell growth and apoptosis induction in FaDu human pharyngeal carcinoma cells. These were investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, Live/Dead cell assay, 4',6-diamidino-2-phenylindole dihydrochloride staining, caspase-3 and caspase-7 activation assay, and immunoblotting in FaDu cells. Acacetin induced FaDu cell death in a dose-dependent manner, with an estimated IC₅₀ value of 41.9 µM, without affecting the viability of L-929 mouse fibroblasts as normal cells. Acacetin treatment resulted in nuclear condensation in the FaDu cells. It promoted the proteolytic cleavage of procaspase-3, -7, -8, and -9 with increasing amounts of the cleaved caspase isoforms in FaDu cells. Acacetin-induced apoptosis in FaDu cells was mediated by the expression of Fas and activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase. Immunoblotting showed downregulation of the anti-apoptotic mitochondrial proteins Bcl-2 and Bcl-xL, but upregulation of the mitochondria-dependent pro-apoptotic proteins Bax and Badin FaDu cells after acacetin treatment. These findings indicate that acacetin inhibits cell proliferation and induces apoptotic cell death in FaDu human pharyngeal carcinoma cells via both the death receptor-mediated extrinsic apoptotic pathway and the mitochondria-mediated intrinsic apoptotic pathway.

• Journal of Acupuncture Research
• /
• v.31 no.1
• /
• pp.111-119
• /
• 2014

Objectives : I investigated whether Bee Venom can synergistically strengthen the cytotoxic effects of NK-92 cells, enhancing the inhibition of the growth of Lung Cancer Cells including A549 and NCI-H460 through induction of death receptor dependent extrinsic apoptosis and NO generation in the Nitro-oxide pathway. Methods : Bee Venom inhibited cell proliferation of A549 or NCI-H460 Human Lung Cancer Cells as well as NK-92 Cells. Moreover, when they were co-punctured with NK cells and concomitantly treated by 3 ${\mu}g/ml$ of Bee Venom, more influence was exerted on inhibition of proliferation of A549 or NCI-H460 Human Lung Cancer Cells than BV or NK cell co-culture alone. Results : The expression of Fas, TNFR2, DR3, DR6 in A549 Lung Cancer Cells was significantly increased by co-culture of NK-92 cells and treatment of 3 ${\mu}g/ml$ of Bee Venom, compared to co-culture of NK-92 cells alone, whereas the expression of Fas, TNFR2, DR6 in NCI-H460 Lung Cancer Cells was significantly increased by co-culture of NK-92 cells, representing no synergistic effects in the co-culture of NK-92 cell and concomitant treatment of 3 ${\mu}g/ml$ of Bee Venom. Coincidently, caspase-8, a expression of pro-apoptotic proteins in the extrinsic apoptosis pathway demonstrated same results as the above. Meanwhile, In NO generation, there is little change of NO generation in co-culture of NK-92 cells with A549 cells as well as the co-culture of NK-92 cell with them and concomitant treatment of 3 ${\mu}g/ml$ of Bee Venom, whereas increase of NO generation was shown in co-culture of NK-92 cells with NCI-H460 cells as well as the co-culture of NK-92 cell with them and concomitant treatment of 3 ${\mu}g/ml$ of Bee Venom, although synergistic effects by Bee Venom was not found. Conclusions : These present data provide that Bee Venom could be useful candidate compounds to enhance lung cancer growth inhibiting ability of NK-92 cells through DR expression and the related apoptosis.

• International Journal of Oral Biology
• /
• v.34 no.1
• /
• pp.7-14
• /
• 2009

Nitric oxide (NO) acts as an intracellular messenger at the physiological level but can be cytotoxic at high concentrations. The cells within periodontal tissues, such as gingival and periodontal fibroblasts, contain nitric oxide syntheses and produce high concentrations of NO when exposed to bacterial lipopolysaccharides and cytokines. However, the cellular mechanisms underlying NO-induced cytotoxicity in periodontal tissues are unclear at present. In our current study, we examined the NO-induced cytotoxic mechanisms in human gingival fibroblasts (HGF). Cell viability and the levels of reactive oxygen species (ROS) were determined using a MTT assay and a fluorescent spectrometer, respectively. The morphological changes in the cells were examined by Diff-Quick staining. Expression of the Bcl-2 family and Fas was determined by RT-PCR or western blotting. The activity of caspase-3, -8 and -9 was assessed using a spectrophotometer. Sodium nitroprusside (SNP), a NO donor, decreased the cell viability of the HGF cells in a dose- and time-dependent manner. SNP enhanced the production of ROS, which was ameliorated by NAC, a free radical scavenger. ODQ, a soluble guanylate cyclase inhibitor, did not block the SNP-induced decrease in cell viability. SNP also caused apoptotic morphological changes, including cell shrinkage, chromatin condensation, and DNA fragmentation. The expression of Bax, a member of the proapoptotic Bcl-2 family, was upregulated in the SNP-treated HGF cells, whereas the expression of Bcl-2, a member of the anti-apoptotic Bcl-2 family, was downregulated. SNP augmented the release of cytochrome c from the mitochondria into the cytosol and enhanced the activity of caspase-8, -9, and -3. SNP also upregulated Fas, a component of the death receptor assembly. These results suggest that NO induces apoptosis in human gingival fibroblast via ROS and the Bcl-2 family through both mitochondrial- and death receptor-mediated pathways. Our data also indicate that the cyclic GMP pathway is not involved in NO-induced apoptosis.

• Korean Journal of Plant Resources
• /
• v.32 no.4
• /
• pp.255-263
• /
• 2019

Glycyrrhizae radix is one of the most frequently prescribed ingredients in Oriental medicine, and Glycyrrhizae radix extract has been shown to exert anti-cancer effects. However, the cellular and molecular mechanisms of programed cell death (apoptosis) by Glycyrrhizae radix are poorly defined. In the present study, it was examined the molecular mechanisms of apoptosis by water extracts of Glycyrrhizae radix (GRW) in human bladder T24 cancer cells. It was found that GRW could inhibit the cell growth of T24 cells in a concentration-dependent manner, which was associated with the induction of apoptotic cell death, as evidenced by the formation of apoptotic bodies, DNA fragmentation and increased populations of annexin-V positive cells. The induction of apoptotic cell death by GRW was connected with an up-regulation of pro-apoptotic Bax protein expression and down-regulation of anti-apoptotic proteins (Bcl-2 and Bcl-xL), and inhibition of apoptosis family proteins (XIAP, cIAP-1 and cIAP-2). In addition, apoptosis-inducing concentrations of GRW induced the activation of caspase-9, an initiator caspase of the mitochondrial-mediated intrinsic pathway, and caspase-3, accompanied by proteolytic degradation of PARP. GRW also induced apoptosis via a death receptor-mediated extrinsic pathway by caspase-8 activation, resulting in the down-regulation of total Bid and suggesting the existence of cross-talk between the extrinsic and intrinsic pathways. Taken together, the present results suggest that GRW may be a potential chemotherapeutic agent for the control of human bladder cancer cells.

• Asian Pacific Journal of Cancer Prevention
• /
• v.17 no.sup3
• /
• pp.125-130
• /
• 2016

Nepeta cataria L. has been used in traditional medicine of some countries. Here the cytotoxic and apoptogenic activity of methanol extracts, n-hexane, dichloromethane, ethyl acetate, n-butanol, and acqueous extracts and the essential oil obtained from the aerial parts of the plant were evaluated with PC3, DU-145 and MCF-7 cell lines. Cell viability, histograms of PI stained fragmented DNA in apoptotic cells and Western blot analysis of proteins involved in the cascade of apoptosis were compared in all samples. Thirty components were identified as volatile, representing 99.7% of essential oil composition after GC-MS analysis of the oil obtained from aerial parts of the N. cataria by hydro-distillation. The major oil components of the essential oil were nepetalactone stereoisomers. Comparing IC50 values showed estrogen receptor positive PC3 cells were more sensitive to the cytotoxic effects of N. cataria in comparison with low hormone-receptor presenting DU-145 cells. Among multiple extracts and essential oils of the plant, only the ethyl acetate extract could significantly decrease cell viability in PC3 cells, in a concentration dependent manner. Ethyl acetate extract of N. cataria treated cells showed a sub-G1 peak in PC3 cells in a concentration dependent manner that indicates the involvement of an apoptotic process in ethyl acetate extract-induced cell death. Western blotting analysis showed that in PC3 cells treated with ethyl acetate (48 h) caspase 3 and PARP were cleaved to active forms. Overall, the results suggest that further analytical elucidation of N. cataria in respect to finding new cytotoxic chemicals with anti-tumor activity is warranted.