• Journal of Life Science
• /
• v.21 no.7
• /
• pp.961-968
• /
• 2011

The formation of reactive lipid aldehydes, 4-hydroxynonenal (HNE) is shown to be derived from fatty acid hydroperoxides through the oxidative process. Among its known effects in cytotoxicity, HNE has been implicated in apoptotic cell death. To delineate its putative role as a potential mediator, we investigated the mechanism by which HNE induces apoptosis of endothelial cells (ECs). The anti-proliferative effects of HNE were tested through MTT assay after exposure to various concentrations ($5\sim15\;{\mu}M$) of HNE. We observed apoptotic bodies with propidium iodide staining, and measured the HNE induction of endothelial apoptosis by flow cytometry assay. We observed that cells exposed to HNE for 24 hr resulted in increased poly(ADP-ribose) polymerase cleavage and up-regulation of Bax. Data on the HNE action strongly indicated the involvement of reactive species, namely, intracellular ROS, nitrite, and peroxynitrite. To obtain evidence on the implication of ROS and peroxynitrite in HNE-induced apoptosis, a ROS scavenger, N-acetylcysteine (NAC), and a peroxynitrite scavenger, penicillamine, were tested. Results clearly indicate that the induction of apoptosis by HNE was effectively inhibited by NAC and penicillamine. Based on the present data, we conclude that the endothelial apoptosis induced by HNE involves both ROS generation and peroxynitrite activity. Our new data could lead to a redefinition of HNE action on apoptosis in ECs.

• Journal of Oral Medicine and Pain
• /
• v.32 no.3
• /
• pp.251-261
• /
• 2007

Bile acids and synthetic its derivatives induced apoptosis in various kinds of cancer cells and anticancer effects. Previous studies have been reported that the synthetic chenodeoxycholic acid (CDCA) derivatives showed apoptosis inducing activity on various cancer cells in vitro. It wasn't discovered those materials have apoptosis induced effects on YD9 human oral squamous carcinoma cells. The present study was done to examine the synthetic bile acid derivatives(HS-1199, HS-1200) induced apoptosis on YD9 cells and such these apoptosis events. We administered them in culture to YD9 cells. Tested YD9 cells showed several lines of apoptotic manifestation such as activation of caspase-3, degradation of DFF, production of poly (ADP-ribose) polymerase(PARP) cleavage(HS-1200 only), DNA degradation(HS-1200 only), nuclear condensation, inhibition of proteasome activity, reduction of mitochondrial membrane potential(HS-1200 only) and the release of cytochrome c and AIF to cytosol. Between two synthetic CDCA derivatives, HS-1200 showed stronger apoptosis-inducing effect than HS-1199. Therefore HS-1200 was demonstrated to have the most efficient antitumor effect. Taken collectively, we demonstrated that a synthetic CDCA derivative HS-1200 induced caspases-dependent apoptosis via mitochondrial pathway in human oral sqauamous carcinoma cells in vitro. Our data therefore provide the possibility that HS-1200 could be considered as a novel therapeutic strategy for human orall squamous carcinoma from its poweful apoptosis-inducing activity.

• Journal of Life Science
• /
• v.29 no.3
• /
• pp.295-302
• /
• 2019

Liquiritigenin (LG) is a chiral flavonoid isolated from the roots of licorice. It exhibits multiple biological activities including anti-oxidant, anti-cancer, and anti-inflammatory effects. In particular though, the anti-cancer activity of LG in oral squamous cell carcinoma has yet to be elucidated, and LG-induced apoptosis in oral squamous cell carcinoma remains poorly understood. In the present study, we tested the role of LG in inducing apoptosis in oral squamous cell carcinoma cells. LG treatment of HN22 cells resulted in a dose-dependent inhibition of cell viability as detected by a 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay. The induction of apoptosis in terms of Annexin V/7-Aminoactinomycin D staining, sub-G1 population, and multi-caspase activity were assessed with a $Muse^{TM}$ Cell Analyzer. Flow cytometric analysis revealed that LG treatment resulted in G2/M arrest in cell cycle progression and downregulation of cyclin B1 and CDC2 expression in a concentration-dependent manner. It also resulted in significant upregulation of p27. In addition, LG was seen to trigger the generation of reactive oxygen species and induce CCAAT/enhancer-binding protein homologous protein and 78-kDa glucose-regulated protein in concentration-dependent upregulation. The LG treatment of HN22 cells led to a loss of mitochondrial membrane potential (${\Delta}{\Psi}m$); it also reduced the levels of anti-apoptotic protein and increased the expression of apoptotic protease activating factor-1, cleaved poly (ADP-ribose)polymerase and Bax. Overall, our results indicate that the pro-apoptotic effects of LG in HN22 cells depend on the activation of both intrinsic and extrinsic signaling pathways. Thus, our results suggest that LG constitutes a natural compound with a potential role as an anti-tumor agent in oral squamous cell carcinoma.

• Journal of Oriental Neuropsychiatry
• /
• v.24 no.4
• /
• pp.419-434
• /
• 2013

Objectives: The object of this study is to observe the cognition and motor function recovery effects of Joojakwhan (JJW), a traditional Korean poly-herbal formula for treating various neuropsychiatric diseases such as dementia, for the mildly stroke rats, with 60 minutes of reperfusion transient middle cerebral artery occlusion (tMCAO). Methods: In the present study, 125, 250 and 500 mg/kg of JJW were orally administered, once per day for 10 continuous days 2 hours after the tMCAO. The body weight changes, infarct sizes under 2% 2, 3, 5-triphenyl tetrazolium chloride (TTC) stain, sensorimotor functions and cognitive motor behavior tests were serially monitored with cerebral caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP)-immunoreactivities and histopathological changes. The effects of tMCAO on sensorimotor functions were evaluated by using of limb placing and body-swing tests, and the cognitive motor behaviors were also observed with water maze tests. Results: From the results of tMCAO, with marked decreases of body weights, disorders of sensorimotor functions increases the limb placing test scores, and decrease the numbers and percentages of body swings to the ipsilateral sides. The cognitive motor behaviors increases the distances and time to reach the escape platform which included the inhibitions of the decreases with repeated trials that were observed with focal cerebral cortex infarct volumes. In addition, the marked increases of the atrophy, numbers of degeneration, caspase-3- and PARP-immunoreactive cells around peri-infarct ipsilateral cerebral cortex were also observed in tMCAO controls when compared with the sham control rats, respectively. Conclusions: The results obtained from this study suggest that oral administrations of JJW indicate obvious cognitions and motor function recoveries of the rats with tMCAO, mild strokes, which are mediated by neuro-protective effects through known antioxidant effects of components.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.5
• /
• pp.648-657
• /
• 2015

H9, a novel herbal extract, demonstrated cytotoxicity in A549 non-small cell lung cancer (NSCLC) cell lines. In this study, we investigated whether H9, and/or co-treatment with an anticancer drug, pemetrexed (PEM), inhibited tumor growth in BALB/c nude mice models bearing A549 NSCLC cells. The mice were separated into groups and administered H9 and PEM for 2 weeks. Protein and mRNA levels were detected using western blotting and reverse transcription polymerase chain reaction, respectively; immunohistochemistry (IHC) was also performed on the tumor tissues. H9 and co-treatment with PEM induced the cleavage of proapoptotic factors, such as caspase-3, caspase-8, caspase-9, and poly(ADP)-ribose polymerase (PARP). Expression levels of cell-death receptors involving Fas/FasL, TNF-related apoptosisinducing ligands (TRAIL), and TRAIL receptors were increased by H9 and co-treatment with PEM. Furthermore, analysis of levels of cell-cycle modulating proteins indicated that tumor cells were arrested in the G1/S phase. In addition, the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt survival signaling pathways were inhibited by H9 and co-treatment with PEM. In conclusion, H9 and co-treatment with PEM inhibited tumor growth in BALB/c nude mice models bearing A549 NSCLC cells. These results indicate that H9 and co-treatment with PEM can be used as an anticancer therapy in NSCLC.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.10
• /
• pp.4369-4376
• /
• 2015

Background: To investigate in-vitro antagonistic effect of low-dose liquiritigenin on gemcitabine-induced capillary leak syndrome (CLS) in pancreatic adenocarcinoma via inhibiting reactive oxygen species (ROS)-mediated signalling pathways. Materials and Methods: Human pancreatic adenocarcinoma Panc-1 cells and human umbilical vein endothelial cells (HUVECs) were pre-treated using low-dose liquiritigenin for 24 h, then added into gemcitabine and incubated for 48 h. Cell viability, apoptosis rate and ROS levels of Panc-1 cells and HUVECs were respectively detected through methylthiazolyldiphenyl-tetrazoliumbromide (MTT) and flow cytometry. For HUVECs, transendothelial electrical resistance (TEER) and transcellular and paracellular leak were measured using transwell assays, then poly (ADP-ribose) polymerase 1 (PARP-1) and metal matrix proteinase-9 (MMP9) activity were assayed via kits, mRNA expressions of p53 and Rac-1 were determined through quantitative polymerase chain reaction (qPCR); The expressions of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and PARP-1 were measured via western blotting. Results: Low-dose liquiritigenin exerted no effect on gemcitabine-induced changes of cell viability, apoptosis rate and ROS levels in Panc-1 cells, but for HUVECs, liquiritigenin ($3{\mu}M$) could remarkably elevate gemcitabine-induced decrease of cell viability, transepithelial electrical resistance (TEER), pro-MMP9 level and expression of ICAM-1 and VCAM-1 (p<0.01). Meanwhile, it could also significantly decrease gemcitabine-induced increase of transcellular and paracellular leak, ROS level, PARP-1 activity, Act-MMP9 level, mRNA expressions of p53 and Rac-1, expression of PARP-1 and apoptosis rate (p<0.01). Conclusions: Low-dose liquiritigenin exerts an antagonistic effect on gemcitabine-induced leak across HUVECs via inhibiting ROS-mediated signalling pathways, but without affecting gemcitabine-induced Panc-1 cell apoptosis. Therefore, low-dose liquiritigenin might be beneficial to prevent the occurrence of gemcitabine-induced CLS in pancreatic adenocarcinoma.

• Journal of Applied Biological Chemistry
• /
• v.57 no.2
• /
• pp.103-111
• /
• 2014

A1E is an extract from traditional Asian medicinal plants that has therapeutic activities against cancers, metabolic disease, and other intractable conditions. However, its mechanism of action on cervical cancer has not been studied. In order to ascertain if A1E would have pronounced anti-cervical cancer effect, cervical cancer cells were incubated with A1E and apoptosis was detected by nuclear morphological changes, annexin V-FITC/PI staining, cell cycle analysis, western blotting, Reverse-transcription polymerase chain reaction, and measurement of mitochondrial membrane potential. Expression of human papiloma virus E6 and E7 oncogenes was down-regulated in A1E-treated cervical cancer cells, while p53 and retinoblastoma protein levels were enhanced. A1E also perturbed cell cycle progression at sub-G1 and altered cell cycle regulatory factors in SiHa cervical cancer cells. A1E activated apoptotic intrinsic pathway markers such as caspase-9, caspase-3 and poly ADP-ribose polymerase, and down-regulated expression of Bcl-2 and Bcl-xl. A1E induced mitochondrial membrane potential collapse and cytochrome c release, and inhibited phosphatidylinositol 3-kinase (PI3K)/Akt, key factors involved in cell survival signaling. Taken all these results, A1E induced apoptosis via activation of the intrinsic pathway and inhibition of the PI3K/Akt survival-signaling pathway in SiHa cervical cancer cells. In conclusion, A1E exerts anti-proliferative action growth inhibition on cervical cancer cells through apoptosis which demonstrates its anti-cervical cancer properties.

• Journal of the Korea Academia-Industrial cooperation Society
• /
• v.20 no.8
• /
• pp.303-312
• /
• 2019

Oxidative stress in kidneys can precede the development of chronic renal injury. We investigated the antioxidative effect of membrane-free stem cell extract (MFSCE) from adipose tissue in LLC-$PK_1$ renal proximal tubule cells. Treatment of LLC-$PK_1$ cells with MFSCE showed the up-regulation of heme-oxygenase-1, thioredoxin reductase 1, and NADPH quinine oxidoreductase-1 protein expressions, which are proteins related with antioxidative activities. When oxidative stress was induced by 3-morpholinosydnonimine (SIN-1), cell viability was decreased, indicating that LLC-$PK_1$ cells were damaged by SIN-1. However, MFSCE significantly elevated cell viability from 58.84% to 64.43% at the concentration of $2.5{\mu}g/mL$ in oxidative stress-induced LLC-$PK_1$ cells. Furthermore, MFSCE ameliorated inflammation and apoptosis in SIN-1-treated LLC-$PK_1$ cells by modulating protein expressions. Inducible nitric oxide synthase and cyclooxygenase-2 protein expressions were down-regulated when LLC-$PK_1$ cells were treated with MFSCE. Apoptosis-related proteins, including B-cell lymphoma-2-associated X protein/B-cell lymphoma-2 ratio, cleaved caspase-3, and cleaved-poly (ADP-ribose) polymerase, were also down-regulated. It indicated that MFSCE protected apoptosis against oxidative stress in LLC-$PK_1$ cells. Taken together, these results suggested that MFSCE had a protective effect against SIN-1-induced oxidative stress in LLC-$PK_1$ cells. Therefore, MFSCE could be a promising therapeutic agent for oxidative stress-induced renal injury.

• Radiation Oncology Journal
• /
• v.19 no.2
• /
• pp.153-162
• /
• 2001

Purpose : The mechanical insights of death of cancer cells by ionizing radiation are not of yet clearly defined. Recent evidences have demonstrated that radiation therapy may induce cell death via activation of signaling pathway for apoptosis in target cells. This study is designed whether ionizing radiation may activate the signaling cascades of apoptosis including caspase family cysteine pretenses, $Bcl_2/Bax$, cytochrome c and Fas/Fas-L in target cells. Materials and Methods : HL-60 cells were irradiated in vitro with 6 MV X-ray at dose ranges from 2 Gy to 32 Gy. The cell viability was tested by M assay and the extent of apoptosis was determined using agarose gel electrophoresis. The activities of caspase proteases were measured by proteolytic cleavages of substrates. Western blot analysis was used to monitor PARP, Caspase-3, Cytochrome-c, Bcl-2, Bax, Fas and Fas-L. Results : Ionizing radiation decreases the viability of HL-60 cells in a time and dose dependent manner. Ionizing radiation-induced death in HL-60 cells is an apoptotic death which is revealed as characteristic ladder-pattern fragmentation of genomic DNA over 16 Gy at 4 hours. ionizing radiation induces the activation of caspase-2, 3, 6, 8 and 9 of HL-60 cells in a time-dependent manner. The activation of caspase-3 pretense is also evidenced by the digestion of poly (ADP-ribose) polymerase and procaspase 3 with 16Gy ionizing irradiation. Anti-apoptotic Bcl2 expression is decreased but apoptotic Bax expression is increased with mitochondrial cytochrome c release in a time- dependent manner. In addiiton, expression of Fas and Fas-L is also increased in a time dependent manner. Conclusion : These data suggest that ionizing radiation-induced apoptosis is mediated by the activation of various signaling pathways including caspase family cysteine proteases, $Bcl_2/Bax$, Fas and Fas-L in a time and dose dependent manner.

• Radiation Oncology Journal
• /
• v.18 no.3
• /
• pp.214-219
• /
• 2000

Purpose : To Investigate the pathways of radiation induced apoptosls and the effect of cysteamine (${\beta}$-mercaptoethyiamine), as a radioprotector, on it. Materials and Methods : HL-50 ceils were assigned to control, irradiated, and cysteamlne (1 mM, 10mM) pretreated groups. Irradiation was given In a single fraction of 10 Gy (6 MV x-ray) and cysteamine was administered 1 hour before irradiation. The activities of caspase-8 were measured in control and irradiated group to evaluate its relation to the radiation Induced apoptosis. To evaluate the role of cysteamine In radiation Induced apoptosis, the number of viable cells, the expression and activity of caspase-3, and the expression of poly (ADP-ribose) polymerase (PARP) were measured and compared after irradiating the HL-60 celis with cysteamine pretreatment or not. Results : The intraceliular caspase-8 activity, known to be related to the death receptor induced apoptosis, was not affected by irradiation(p>0.05). The number of viable cells began to decrease from 6 hours after irradiation (p>0.05), but the number of viable cells In 1 mM cysteamine pretreated group was not decreased after irradiation and was similar to those in the control group. In caspase-3 analyses, known as apoptosis executioner, its expression was not different but its activity was Increased by irradiation(p>0.05). However, this Increase of activity was suppressed by the pretreatment of 1 mM cysteamine. The cleavage of PARP, thought to be resulted from caspase-3 activation, occurred after irradiation which was attenuated by the pretreatment of 1 mM cysteamine. Conclusion : These results show that radiation induced apoptotic process is somewhat different from death receptor induced one and the pretreatment of 1 mM cysteamine has a tendency to decrease the radiation-induced apoptosis in HL-60 cells.