• Journal of Photoscience
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• v.9 no.2
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• pp.229-232
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• 2002

There are two distinct UV-responsive signaling pathways in UV-irradiated mammalian cells, i.e., the DNA damage-dependent and -independent pathways. The former occurs in nucleus and results in growth arrest and apoptosis via post-translational modification of p53. The latter is initiated by oxidative stress and/or by damages in cell membrane or cytoplasm, which activate signaling cascade through intracellular molecules including mitogen activated protein kinases (MAPK). In normal human fibroblastic cells, all of MAPK family members, extracellular signal-related kinases (ERK), c-Jun N-terminal kinases (JNK) and p38, were rapidly phosphorylated following UV-irradiation. ERK phosphorylation was suppressed by an inhibitor of receptor tyrosine kinases (RTK). As ERK usually responds to mitogenic stimuli from RTK ligands, UV-induced ERK phosphorylation may be linked to the proliferation of survived cells. In contrast, phosphorylation of JNK and p38, as well as apoptosis, were modulated by the level of UV-generated oxidative stress Therefore, JNK and p38 may take part in oxidative stress-mediated apoptosis. Phosphorylation of p53 at Ser and Thr residues are essential for stabilization and activation of p53. Among several sites reported, we confirmed phosphorylation at Ser-15 and Ser-392 after UV-irradiation. Both of these were inhibited by a phosphoinositide 3-kinase inhibitor, presumably due to the shutdown of signals from DNA damage to p53. Phosphorylation at Ser-392 was also sensitive to an antioxidant and a p38 inhibitor, suggesting that Ser-392 of p53 is one of the possible points where DNA damage-dependent and -independent apoptic signals merge. Thus, MAPK pathway links UV-induced intracellular signals to the nuclear responses and modifies DNA damage-dependent cellular outcome, resulting in the determination of cell death.

• Journal of Photoscience
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• v.9 no.2
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• pp.482-484
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• 2002

UV irradiation activates various intracellular signaling pathways causing cell death in a DNA damage-dependent and an independent manner. As DNA photoproducts, major forms of DNA damage, are maximally formed by UV light at 260-nm, short wavelength UV (UVC) is more harmful than middle wavelength UV (UVB). However, the differences or similarities in responses of DNA damage-independent intracellular signaling molecules to UVB and UVC are not elucidated. We examined activation of signaling molecules towards apoptosis in normal human fibroblastic cells after irradiation with UVB or UVC at a dose generating the equal amount of DNA photoproducts. Both UVB and UVC induced transient phosphorylation of ERK and sustained phosphorylation of p38. Phosphorylation of p53 at Ser15 and at Ser392 residues were also observed, which were inhibited by a phosphoinositide 3-kinase inhibitor, wortmannin. In contrast, an antioxidant N-acetyl-cysteine and a p38 inhibitor SB203580 suppressed only Ser392 phosphorylation, suggesting that UV-induced oxidative stress and p38 activation were involved in the phosphorylation of this site. The apoptic signals such as mitochondrial cytochrome C release and annexin V binding were then observed. Overall, no difference was found in chronological responses of p53, MAPK, and apoptosis between UVB-irradiated and UVC-irradiated cells. These results suggested that DNA damage-independent intracellular signaling molecules similarly responded to UVB and UVC when the equal level of DNA photoproducts were generated.

• Nutrition Research and Practice
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• v.2 no.4
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• pp.322-325
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• 2008

The aim of present study was to investigate the effects of kaempferol on cellular proliferation and cell cycle arrest and explore the mechanism for these effects in human breast carcinoma MDA-MB-453 cells. Cells were treated with kaempferol at various concentrations (ranging from 1 to $200\;{\mu}M$) for 24 and 48 hrs. Kaempferol significantly inhibited cancer cell growth in cells exposed to 50 and $10\;{\mu}M$ of kaempferol and incubated for 24 and 48 hrs, respectively. Exposure to kaempferol resulted in cell cycle arrest at the G2/M phase. Of the G2/M-phase related proteins, kaempferol down-regulated CDK1 and cyclin A and B in cells exposed to kaempferol. In addition, small DNA fragments at the sub-G0 phase were increased by up to 23.12 and 31.90% at 10 and $50\;{\mu}M$ incubated for 24 and 48 hrs, respectively. The kaempferol-induced apoptosis was associated with the up-regulation of p53. In addition, the phosphorylation of p53 at the Ser-15 residue was observed with kaempferol. Kaempferol inhibits cell proliferation by disrupting the cell cycle, which is strongly associated with the induction of arrest at G2/M phase and may induce apoptosis via p53 phosphorylation in human breast carcinoma MDA-MB-453 cells.

• Journal of Microbiology and Biotechnology
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• v.25 no.3
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• pp.418-422
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• 2015

In the course of screening for novel cell cycle inhibitors and apoptotic inducers, CR389, elucidated as 5-(1H-benzoimidazol-2-yl)-1H-pyridin-2-one, was generated as a new hit compound. Flow cytometric analysis and western blots of PA-1 cells treated with $60{\mu}M$ CR389 revealed an appreciable cell cycle arrest at the G2/M phase through direct inhibition of the CDK1 complex. In addition, activation of p53 via phosphorylation at Ser15 and subsequent up-regulation of p21^CIP1 showed that CR389 also induces p53-dependent-p21^CIP1-mediated cell cycle arrest. Furthermore, apoptotic induction in $60{\mu}M$ CR389-treated PA-1 cells is associated with the release of cytochrome c from mitochondria through up-regulation of the proapoptotic Bax protein, which results in the activation of procaspase-9 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP). Accordingly, CR389 seems to have multiple mechanisms of antiproliferative activity through p53-mediated pathways against human ovarian cancer cells. Therefore, we conclude that CR389 is a candidate therapeutic agent for the treatment of human ovarian cancer via the activation of p53.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.287-294
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• 2016

The effect of kaempferol (3,5,7,4-tetrahydroxyflavone), a flavonoid compound that was identified in barnyard millet (Echinochloa crus-galli var. frumentacea) grains, on G₂-checkpoint and apoptotic pathways was investigated in human acute leukemia Jurkat T cell clones stably transfected with an empty vector (J/Neo) or a Bcl-xL expression vector (J/Bcl-xL). Exposure of J/Neo cells to kaempeferol caused cytotoxicity and activation of the ATM/ATR-Chk1/Chk2 pathway, activating the phosphorylation of p53 (Ser-15), inhibitory phosphorylation of Cdc25C (Ser-216), and inactivation of cyclin-dependent kinase 1 (Cdk1), with resultant G₂-arrest of the cell cycle. Under these conditions, apoptotic events, including upregulation of Bak and PUMA levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9, -8, and -3, anti-poly (ADP-ribose) polymerase (PARP) cleavage, and accumulation of apoptotic sub-G₁ cells, were induced without accompanying necrosis. However, these apoptotic events, except for upregulation of Bak and PUMA levels, were completely abrogated in J/Bcl-xL cells overexpressing Bcl-xL, suggesting that the G₂-arrest and the Bcl-xL-sensitive mitochondrial apoptotic events were induced, in parallel, as downstream events of the DNA-damage-mediated G₂-checkpoint activation. Together these results demonstrate that kaempferol-mediated antitumor activity toward Jurkat T cells was attributable to G₂-checkpoint activation, which caused not only G₂-arrest of the cell cycle but also activating phosphorylation of p53 (Ser-15) and subsequent induction of mitochondria-dependent apoptotic events, including Bak and PUMA upregulation, Bak activation, Δψm loss, and caspase cascade activation.

• Journal of Life Science
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• v.23 no.10
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• pp.1230-1238
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• 2013

The regulatory effect of the tumor-suppressor protein p53 on the apoptogenic activity of $17{\alpha}$-estradiol ($17{\alpha}-E_2$) was compared between HCT116 ($p53^{+/+}$) and HCT116 ($p53^{-/-}$) cells. When the HCT116 ($p53^{+/+}$) and HCT116 ($p53^{-/-}$) cells were treated with $2.5{\sim}10{\mu}M$ $17{\alpha}-E_2$ for 48 h or with $10{\mu}M$for various time periods, cytotoxicity and an apoptotic sub-$G_1$ peak were induced in the HCT116 ($p53^{+/+}$) cells in a dose- and time-dependent manner. However, the HCT116 ($p53^{-/-}$) cells were much less sensitive to the apoptotic effect of $17{\alpha}-E_2$. Although $17{\alpha}-E_2$ induced aberrant mitotic spindle organization and incomplete chromosome congregation at the equatorial plate, $G_2/M$ arrest was induced to a similar extent in both cell types. In addition, $17{\alpha}-E_2$-induced activation of Bak and Bax, ${\Delta}{\Psi}m$ loss, and PARP degradation were more dominant in the HCT116 ($p53^{+/+}$) than in the HCT116 ($p53^{-/-}$) cells. In accordance with enhancement of p53 phosphorylation (Ser-15) and p53 levels, p21 and Bax levels were elevated in the HCT116 ($p53^{+/+}$) cells treated with $17{\alpha}-E_2$. The HCT116 ($p53^{-/-}$) cells exhibited barely or undetectable levels of p21 and Bax, regardless of $17{\alpha}-E_2$ treatment. On the other hand, although the level of Bcl-2 was slightly lower in the HCT116 ($p53^{+/+}$) than in the HCT116 ($p53^{-/-}$) cells, it remained relatively constant after the $17{\alpha}-E_2$ treatment. Together, these results show that among the components of the $17{\alpha}-E_2$-induced apoptotic-signaling pathway, which proceeds through mitotic spindle defects causing mitotic arrest, subsequent activation of Bak and Bax and the mitochondria-dependent caspase cascade, leading to PARP degradation, $17{\alpha}-E_2$-induced activation of Bak and Bax is the upstream target of proapoptotic action of p53.

• Biomedical Science Letters
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• v.13 no.2
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• pp.75-81
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• 2007

Sodium salicylate (NaSal), a chemopreventive drug, has been shown to induce apoptosis and cell circle arrest depending on its concentrations in a variety of cancer cells. In A549 cells, low concentration of NaSal (5$\sim$10 mM) induces cell cycle arrest, whereas it induces apoptosis at higher concentration of 20 mM. In the present study, we examined the molecular mechanism for NaSal-induced cell cycle arrest. NaSal induced expression of p53, p21 (Wafl/Cipl), and p27 (Kipl) that play important roles in cell cycle arrest. p53 induction was mediated by its phosphorylation at Ser-15 that could be prevented by the PI3K-related kinase (ATM, ATR and DNA-PK) inhibitors including wortmannin, caffeine and LY294002. In addition, NaSal-induction of p2l (Wafl/Cipl) was detected in P53 (+/+) wild type A549 cells but not in p53 (-/-) mutant H1299 cells, indicating p53-dependent p21 (Wafl/Cipl) induction. In contrast, p27 (Kipl) that is a negative regulate. of cell cycle with p21 (Wafl/Cipl) was observed both in A549 cells and H1299 cells. Thus, 5 mM NaSal appeared to cause cell cycle arrest through inducing the cyclin-dependent kinase inhibitor p21 (Wafl/Cipl) via PI3K-related protein kinase-dependent p53 activation as well as by up-regulating p27 (Kipl) independently of p53 in A549 cells.

• BMB Reports
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• v.41 no.10
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• pp.716-721
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• 2008

To explore the effects of deregulated expression of the EBNA1 binding protein 2 (EBP2) on cell growth, we generated human HEK293 stable clones constitutively expressing an EBP2-EGFP fusion protein. We found both RNA and protein levels of cyclin E1, a dominant oncoprotein, were elevated in the EBP2- EGFP stable clones. These findings were confirmed by flow cytometry bivariate analysis of cyclin expression versus DNA content. Moreover, the increase in p21 expression and the specific phosphorylation at Ser1981 of ATM and Ser15 of p53 were also observed in these stable clones, and these observations may explain the failure to observe an increase in Cdk2 kinase activity. In addition, after one year of passage culture, the EBP2-EGFP stable clones tended to lose 4 to 5 chromosomes per cell when compared to that of control cells. All of these findings provide a possible link between deregulated expression of EBP2 and tumor development.

• Natural Product Sciences
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• v.23 no.1
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• pp.21-28
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• 2017

In our program to search for new AMP-activated protein kinase (AMPK) activators from plants that exert potential anticancer property, we found that an EtOAc extract of Myristica fragrans (nutmeg) activated AMPK enzyme in human breast cancer MCF-7 cells. Two major diarylbutane-type lignans, macelignan and meso-dihydroguaiaretic acid (MDGA), were isolated as active principles from this extract. Treatment of breast cancer cells with two compounds induced cellular apoptosis, evidenced by cleavage of poly-(ADP-ribose) polymerase (PARP) and Ser 15 phosphorylation of p53. Moreover, macelignan and MDGA significantly inhibited the colony formation of MCF-7 breast cancer cells on soft agar. Intraperitoneal injection of macelignan and MDGA (20 mg/kg) suppressed the tumor growth of 4T1 mammary cancer cells. These results indicate that the chemopreventive effects of two major diarylbutane-type lignans from Myristica fragrans (nutmeg) may be associated with induction of apoptosis presumably through AMPK activation.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1591-1595
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• 2012

We previously isolated a novel compound, HY251, with the molecular structure of 3-propyl-2-vinyl-1,2,3,3a,3b,6,7,7a,8,8a-decahydrocyclopenta[a]indene-3,3a,7a,8a-tetraol from the roots of Aralia continentalis. The current study was designed to evaluate the detailed molecular mechanisms underlying the apoptotic induction by HY251 in human ovarian cancer PA-1 cells. TUNEL assay and Western blot analyses revealed an appreciable apoptotic induction in PA-1 cells treated with $60{\mu}M$ of HY251 for 24 h. This apoptotic induction was associated with caspase-8-dependent Bid cleavage, which in turn resulted in the formation of pro-apoptotic truncated Bid (tBid), and activation of caspase-9 and -3, as well as the cleavage of poly(ADP-ribose) polymerase (PARP). Moreover, we found that this death event was also associated with the significant up-regulation and activation of the p53 tumor-suppressor protein through phosphorylation at Ser15. Therefore, we suggest that HY251 may be a potent cancer chemotherapeutic candidate for the treatment of ovarian cancer.