• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.3
• /
• pp.1285-1290
• /
• 2014

The aim was to determine whether ultrasound targeted microbubble destruction (UTMD) promotes dual targeting of HSP72 and HSC70 for therapy of castration-resistant prostate cancer (CRPC), to improve the specific and efficient delivery of siRNA, to induce tumor cell specific apoptosis, and to find new therapeutic targets specific of CRPC.VCaP cells were transfected with siRNA oligonucleotides. HSP70, HSP90 and cleaved caspase-3 expression were determined by real-time quantitative polymerase chain reaction and Western blotting. Apoptosis and transfection efficiency were assessed by flow cytometry. Cell viability assays were used to evaluate safety. We found HSP72, HSC70 and HSP90 expression to be absent or weak in normal prostate epithelial cells (RWPE-1), but uniformly strong in prostate cancerous cells (VCaP). UTMD combined with dual targeting of HSP72 and HSC70 siRNA improve the efficiency of transfection, cell uptake of siRNA, downregulation of HSP70 and HSP90 expression in VCaP cells at the mRNA and protein level, and induction of extensive tumor-specific apoptosis. Cell counting kit-8 assays showed decreased cellular viability in the HSP72/HSC70-siRNA silenced group. These results suggest that the combination of UTMD with dual targeting HSP70 therapy for PCa may be most efficacious, providng a novel, reliable, non-invasive, safe targeted approach to improve the specific and efficient delivery of siRNA, and achieve maximal effects.

• The Korea Journal of Herbology
• /
• v.21 no.2
• /
• pp.37-46
• /
• 2006

Objectives : The purpose of this study was to investigate the effect of Dangguibohyultang (DB) and its combination (DB-I; Astragali membraneus BUNGE : Angelica gigas NAKAI=5:1, DB-II; Astragali membraneus BUNGE:Angelica gigas NAKAI=1:1, DB-III; Astragali membraneus BUNGE:Angelica gigas NAKAI=1:5,) on apoptosis in human colorectal adenocarcinoma HCT116 cells. Methods : To study the cytotoxic effect of methanol extract of DB-I, DB-II and DB-III on HCT116 cells, the cell viability was determined by XTT reduction method and ttypan blue exclusion assay. To confirm the induction of apoptosis, the cleavage of poly ADP-ribose polymerase (PARP), a substrate for caspase-3 and a typical sign of apoptosis, and the activation of procaspase-3, -8 and -9 were examined by western blot analysis. Furthermore, DB-induced apoptosis was confirmed by DNA fragmentation. The release of cytochrome C from mitochondria to cytosol, the level of Bcl-2 and Bax, and the expressions of Raf/MEK/ERK were examined by western blot analysis. Results : DB-I and DB-II reduced proliferation of HCT116 cells in a dose-dependent manner. DB-I and DB-II decreased procaspase-3, -8, -9 levels in a dose-dependent manner and induced the clevage of PARP. DB-I and DB-II also triggered the mitochondrial apoptotic signaling by increasing the release of cytochrome C from mitochondria to cytosol, decreasing of anti-apoptotic Bcl-2, and increasing of pro-apoptotic Bax. DB-I and DB-II decreased the activation of Ras/Raf/MEK/ERK cascade in a dose-dependent manner. Conclusion : These results suggest that DB-I and DB-II induce apoptosis via mitochondrial pathway in HCT116 cells. Furthermore, Raf/MEK/ERK cascade is involved in DB-induced apoptosis. These results suggest that DB is potentially useful as a chemotherapeutic agent in human liver cancer.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.4
• /
• pp.1711-1714
• /
• 2014

Objective: To explore the effect of Withaferin A on A549 cellular proliferation and apoptosis in non-small cell lung cancer (NSCLC). Materials and Methods: NSCLC cell line A549 was selected to explore the effect of Withaferin A on A549 cellular proliferation, apoptosis and the PI3K/Akt signal pathway capable of regulating tumor biological behavior by assessment of cellular proliferation, cellular apoptotic rates and cellular cycling as well as by immuno-blotting. Results: Withaferin A could inhibit A549 cellular proliferation and the control rate was dosage-dependent (P<0.05), which also increased time-dependently with the same dosage of Withaferin A (P<0.05). The apoptotic indexes in A549 cells treated with 0, 2.5, 5.0, 10.0 and 20.0 ${\mu}mol{\cdot}L^{-1}$ Withaferin A for 48 h were significantly different (P<0.05). In addition, the apoptotic rates of each group in both early and advanced stages were higher than those in 0 ${\mu}mol{\cdot}L^{-1}$ (P<0.05), which were evidently higher after 48 h than those after 24 h (P<0.05). A549 cells treated by Withaferin A for 48 h were markedly lower in Bcl-2 level and obviously higher in Bax and cleaved caspase-3 levels than those treated by 0 ${\mu}mol{\cdot}L^{-1}$ Withaferin A (P<0.05), and there were significant differences among 5, 10 and 20 ${\mu}mol{\cdot}L^{-1}$ Withaferin A (P<0.05). The ratios of A549 cells treated by Withaferin A for 48 h in G0/G1 stage were higher than those in 0 ${\mu}mol{\cdot}L^{-1}$, while those in S and G2/M stages were obviously lower than those in G2/M stage, and there were significant differences in 5.0, 10.0 and 20.0 ${\mu}mol{\cdot}L^{-1}$ Withaferin A (P<0.05). Additionally, p-Akt/Akt values were in reverse association with dosage, and the differences were significant (P<0.05). Conclusion: Withaferin A can inhibit the proliferation and apoptosis of A549 cells by suppressing activation of the PI3K/Akt pathways.

• The Journal of Internal Korean Medicine
• /
• v.34 no.2
• /
• pp.178-191
• /
• 2013

Objectives : In this study, we made an effort to investigate the protective mechanism of Ukgan-san (UGS) extracts on hypoxia-induced C6 glial cell death. Methods : The cell viability was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MMT) assay and cell morphological changes were analysed with microscope after staining with crystal violet (CV). Reactive oxygen species (ROS) formation was assessed by flow cytometer after staining with 2'7'-dichlorofluorescein diacetate (DCF-DA). We also analyzed expression of hypoxia-inducible factor-1 alpha (HIF-$1{\alpha}$) and p53, processing of procaspase-3 and procyclic acidic repetitive protein (PARP) by western blot method. Results : We estimated the elevated cell viability by UGS extract on $CoCl_2$-induced C6 glial cells. UGS attenuated $CoCl_2$-induced ROS formation in C6 glial cells and also showed a protective activity compared to antioxidants and exhibited abrogation of LDH-released by $CoCl_2$. UGS suppressed the typical apoptotic cell death markers, caspase-3 and PARP activation. UGS inhibited $CoCl_2$-induced HIF-1${\alpha}$ expression which is known as a major regulator for hypoxia-induced cell death, and suppressed p53 expression. Conclusions : These results suggest that UGS extract contains protective constituents for hypoxia-induced C6 glial cell death.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.35 no.3
• /
• pp.143-154
• /
• 2013

Purpose: This study was to find efficacy of integrin alpha2 (${\alpha}_2$) and epidermal growth factor receptor (EGFR) as tumor marker of oral squamous cell carcinoma (SCC) and clarify the selective cell death effect of anti-integrin ${\alpha}_2$ and anti-EGFR on SCC cells, additionally testify conjugated gold nanoparticles (GNP) with air plasma for selective cell death of oral SCC. Methods: Expression of integrin ${\alpha}_2$, EGFR on human SCC cells (SCC25) were examined by western blot. SCC25 cells were treated with anti-integrin ${\alpha}_2$, anti-EGFR and analysed by Hemacolor staining, immunoflorescence staining, FACS flow cytometry. Conjugated GNP with integrin ${\alpha}_2$, EGFR antibody were treated by air plasma on SCC cells. Results: Integrin ${\alpha}_2$ and EGFR were over-expressed on SCC25 cells than normal lung WI-38 cells. The cell viability rate of SCC25 cells treated with anti-integrin ${\alpha}_2$, anti-EGFR was lower than WI-38 cells. The concentration changes of nucleus, releasing cytochrome c and apoptosis inducing factor (AIF) from mitochondria to cytosol were observed. The changes of proteins related with apoptosis were observed. Increase of bax, bcl-xL, activation of caspase-3, -7, -9, and fragmentation of PARP, DFF45 and decrease of lamin A/C in SCC25 cells were observed. In FACS, increase of sub-$G_1$ and S phase was observed. Cell cycle related proteins, Such as cyclin D1, cyclin dependent kinase (CDK) 4, cyclin A, cyclin E, CDK 2, p27 were decreased. After SCC25 cells treated with conjugatged GNP-Integrin ${\alpha}_2$, GNP-EGFR, additionally air plasma, the cell death rate was significantly increased. Conclusion: Integrin ${\alpha}_2$, EGFR were over-expressed in oral SCC cells. Anti-integrin ${\alpha}_2$, anti-EGFR in SCC25 cells induced apoptosis selectively. When GNP-anti integrin ${\alpha}_2$, GNP-anti EGFR were treated with air plasma on SCC25 cells, cancer cells were died more selectively. GNP-anti integrin ${\alpha}_2$, GNP-anti EGFR with air plasma could be treatment choice of oral SCC.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.11
• /
• pp.1355-1363
• /
• 2009

In the present study, the neuroprotective effects of astaxanthin on $H_2O_2$-mediated apoptotic cell death, using cultured mouse neural progenitor cells (mNPCs), were investigated. To cause apoptotic cell death, mNPCs were pretreated with astaxanthin for 8 h and followed by treatment of 0.3 mM $H_2O_2$. Pretreatment of mNPCs with astaxanthin significantly inhibited $H_2O_2$-mediated apoptosis and induced cell growth in a dose-dependent manner. In Western blot analysis, astaxanthin-pretreated cells showed the activation of p-Akt, p-MEK, p-ERK, and Bcl-2, and the reduction of p-P38, p-SAPK/JNK, Bax, p-GSK3b, cytochrome c, caspase-3, and PARP. Because $H_2O_2$ triggers caspases activation, this study examined whether astaxanthin can inhibit caspases activation in $H_2O_2$-treated mNPCs. After $H_2O_2$ treatment, caspases activities were prominently increased, but astaxanthin pretreatment significantly inhibited $H_2O_2$-mediated caspases activation. Astaxanthin pretreatment also significantly recovered the ATP production ability of $H_2O_2$-treated cells. These findings indicate that astaxanthin inhibits $H_2O_2$-mediated apoptotic features in mNPCs. Inhibition assays with SB203580 ($10\;{\mu}M$, a specific inhibitor of p38) and PD98059 ($10\;{\mu}M$, a specific inhibitor of MEK) clearly showed that astaxanthin can inhibit $H_2O_2$-mediated apoptotic death via modulation of p38 and MEK signaling pathways.

• Asian-Australasian Journal of Animal Sciences
• /
• v.31 no.8
• /
• pp.1325-1335
• /
• 2018

Objective: This study investigated whether spermine supplementation could regulate cell cycle, apoptosis, and amino acid transporter-related genes expression in the thymus and spleen of early weaned piglets. Methods: Eighty female piglets were randomly distributed to receive adequate nutrients supplemented with spermine (0.4 mmol/kg body weight/24 h) or to be provided with restricted nourishment supplemented with normal saline for 7 h or 3, 6, or 9 d in pairs. Results: Regardless of administration time, spermine supplementation significantly up-regulated cyclin A2 gene expression but down-regulated p21 and cyclin D3 mRNA levels in the thymus and spleen and reduced cyclin E2 gene expression in the thymus of piglets (p<0.05). Irrespective of the treatment period, the reduced Bax and caspase-3 gene expressions and improved Bcl-2 mRNA level were observed in the thymus and spleen of spermine-administrated piglets (p<0.05). Regardless of supplementation time, spermine intake significantly enhanced the expressions of amino acid transporter-related genes (SLC1A1, SLC1A5, SLC7A1, SLC7A7, and SLC15A1) in both thymus and spleen, as well as SLC7A9 in the spleen of piglets (p<0.05). In addition, extended spermine administration also markedly promoted cell proliferation, depressed apoptosis and modulated amino acid transport (p<0.05), and such effects were the greatest during prolonged spermine supplementation (6 d) compared to the other time periods (p<0.05). Conclusion: Spermine supplementation may regulate cell cycle during the G1/S phase, suppress apoptosis and modulate amino acid transport. A period of 6 d of spermine supplementation is required to produce the optimal effects on nutritional implications.

• Journal of Ginseng Research
• /
• v.28 no.1
• /
• pp.39-44
• /
• 2004

The present study is to determine whether the Red-ginseng extract has a proliferative or cytotoxic effect on the human neuronal stem cells(hNSCs). The hNSCs were grown and incubated with different doses of Red-ginseng extract. We tested the proliferative or cytotoxic effects by MTT and FACS analysis. Cell viability cell cycle analysis, DNA fragmentation, and bax or PARP expressions were evaluated. The hNSCs showed a proliferafe trend with its peak concentration at 0.3 $\mu\textrm{g}$/$m\ell$. Beyond this point, higher doses decreased viabilities and showed a cytotoxic effect at 10 $\mu\textrm{g}$/$m\ell$. There was a tendency of increased S and G2/M phases during cell proliferation. In a cytotoxic condition, decreased S phase and increased G0/G1 phases were noted, suggesting cell cycle arrest. The cytotoxic effect was associated with increase DNA fragmentation in a dose-dependent manner, However PARP cleavage or bax expression was not detected. Our results suggest that Red-ginseng extract has dual effects, the cell proliferative or cytotoxic effect, on hNSCs in vitro with dose-dependent manner.

• Journal of Ginseng Research
• /
• v.41 no.3
• /
• pp.392-402
• /
• 2017

Background: Previously, we reported that Korean Red Ginseng inhibited liver fibrosis in mice and reduced the expressions of fibrogenic genes in hepatic stellate cells (HSCs). The present study was undertaken to identify the major ginsenoside responsible for reducing the numbers of HSCs and the underlying mechanism involved. Methods: Using LX-2 cells (a human immortalized HSC line) and primary activated HSCs, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assays were conducted to examine the cytotoxic effects of ginsenosides. $H_2O_2$ productions, glutathione contents, lactate dehydrogenase activities, mitochondrial membrane permeabilities, apoptotic cell subpopulations, caspase-3/-7 activities, transferase dUTP nick end labeling (TUNEL) staining, and immunoblot analysis were performed to elucidate the molecular mechanism responsible for ginsenoside-mediated cytotoxicity. Involvement of the AMP-activated protein kinase (AMPK)-related signaling pathway was examined using a chemical inhibitor and small interfering RNA (siRNA) transfection. Results and conclusion: Of the 11 ginsenosides tested, 20S-protopanaxadiol (PPD) showed the most potent cytotoxic activity in both LX-2 cells and primary activated HSCs. Oxidative stress-mediated apoptosis induced by 20S-PPD was blocked by N-acetyl-$\text\tiny L$-cysteine pretreatment. In addition, 20S-PPD concentration-dependently increased the phosphorylation of AMPK, and compound C prevented 20S-PPD-induced cytotoxicity and mitochondrial dysfunction. Moreover, 20S-PPD increased the phosphorylation of liver kinase B1 (LKB1), an upstream kinase of AMPK. Likewise, transfection of LX-2 cells with LKB1 siRNA reduced the cytotoxic effect of 20S-PPD. Thus, 20S-PPD appears to induce HSC apoptosis by activating LKB1-AMPK and to be a therapeutic candidate for the prevention or treatment of liver fibrosis.

• Korean Journal of Plant Resources
• /
• v.23 no.2
• /
• pp.165-171
• /
• 2010

Oxidative stress induced by reactive oxygen intermediates has been implicated in a variety of human diseases including neurodegenerative disorders, cancer, cardiovascular and respiratory diseases, and mode of action of environmental toxicants. Tert-butylhydroperoxide (t-BHP) is an organic lipid hydroperoxide analogue, which is commonly used as a pro-oxidant for evaluating mechanisms involving oxidative stress in cells and tissues. In this study, the underlying mechanisms involved in the protective effects of Hwabuk 94 kiwifruit (Actinidia deliciosa cv. 'Hwabuk 94'), which is cultivated in Jeju, on the t-BHP-induced cytotoxicity in PC12 cell. The pretreatment of rat pheochromocytoma cell line PC12 with Hwabuk 94 extract ($1-100\;{\mu}g/ml$) resulted in a significant recovery from t-BHP-induced cell death and increased Bcl-2 and procaspase-3 expression, whereas the expression of Bax and cleaved PARP were decreased in a dose-dependent manner compared to the control. Furthermore, Hwabuk 94 inhibited the t-BHP-induced p38 MAP kinase and extracellular signal-regulated kinase 1/2, but not c-Jun N-terminal kinase activations. Finally, these findings suggest that Hwabuk 94 kiwifruit might attenuate t-BHP-induced PC12 cell cytotoxicity, at least in part, through the inhibition of signaling pathways mediated by the ERK1/2 and p38 MAP kinase.