Glycyrol Induces Apoptosis in Human Jurkat T Cell Lymphocytes via the Fas-FasL/Caspase-8 Pathway

• Abstract
• Introduction
• Materials and Methods
• Preparation of glycyrol
• Chemicals and reagents
• Cell culture
• Cell viability
• DNA fragmentation analysis
• Flow cytometric analysis
• Caspase-3 activity assay
• Fluorescence microscopy
• Western immunoblot analysis
• Reverse transcriptase PCR (RT‐PCR)
• Statistics
• Supporting information
• Results and Discussion
• Acknowledgements
• References

Abstract

Glycyrrhiza uralensis (Leguminosae) has long been used to treat inflammatory ailments, such as gastric ulcers, arthritis, and rheumatism. From this traditional herbal plant, glycyrol, a coumestan with anti-bacterial and anti-inflammatory activities, was first isolated and synthesized to test its apoptosis-inducing properties in human Jurkat cells. Flow cytometry analysis indicated that glycyrol can arrest the cell cycle in S phase and subsequently induce apoptosis in both time- and dose-dependent manners. Consequently, it was shown that caspase-8 and -9 were involved in the activation of apoptosis after glycyrol treatment. Despite its known NF-κB inhibitory activity, glycyrol did not influence the prosurvival Bcl-2 and the proapoptotic Bax. Interestingly, glycyrol was revealed to enhance the Fas level independently from p53, which even slightly decreased. Thus, glycyrol acts in a similar manner as known cytostatic drugs and may have a potential as lead for the development of drugs for cancer treatment.

Introduction

Apoptosis, or programmed cell death, is controlled by a wide range of necessary death stimuli within (intrinsic pathway) and out of cells (extrinsic pathway) to maintain homeostasis [1], [2]. Disruption of apoptosis leads to a variety of human pathologies including cancer, autoimmune diseases, and neurodegenerative disorders [3], [4]. Intrinsic signaling pathways which involve a diverse array of non-receptor mediated stimuli, for instance oncogene activation or DNA damage within the cell are mitochondrial-initiated events. Intrinsic pathways produce intracellular signals that cause changes in the inner mitochondrial permeability transition (MPT) pore, loss of the mitochondrial transmembrane potential, and release of normally sequestered pro-apoptotic proteins from the intermembrane space into the cytosol. Some of the pro-apototic proteins such as cytochrome c activate the caspase-dependent mitochondrial pathway and others, including caspase activated DNAse (CAD), lead to oligonucleosomal DNA fragmentation and a more pronounced chromatin condensation in the later phase of apoptosis [5]. On the other side, the extrinsic pathway is initiated by ligation of an extracellular death receptor (member of the tumor necrosis factor [TNF] receptor family, such as Fas or TNF receptor-1 [TNFR1]) that contain an intracellular death domain, which can recruit and activate caspase-8 through the Fas-associated death domain adaptor protein (FADD), forming the death-inducing signaling complex (DISC), which once activated cleaves and activates the effector, caspase-3, that irreversibly commits a cell to death [6], [7], [8]. In some cells, the extrinsic pathway intersects the intrinsic pathway through caspase-8 cleavage-mediated activation of the pro-apoptotic BH3-only protein Bid, promoting further caspase activation (caspase-9 and effector caspases, caspase-3, -6, and -7) through the intrinsic pathway [9]. Glycyrol, a prenylated coumestan previously known to exert anti-bacterial [10] and anti-inflammatory [11] activities, was tested for its apoptotic activity in human Jurkat cells. In this report, we first demonstrated the mode of action of glycyrol inducing apoptotic cell death in human Jurkat cells through the direct activation of the Fas-FasL/caspase-8, intersecting the intrinsic pathway independent of p53.

Materials and Methods

Preparation of glycyrol

Glycyrol (purity based on HPLC analysis, 99 %) ([Fig. 1 A]) was first isolated from the root of Glycyrriza uralensis and completely synthesized (purity 99 %) as previously described [12].

Chemicals and reagents

Unless otherwise indicated, all the chemicals were purchased from Sigma-Aldrich Co. The antibodies against Fas, FasL, caspase-8, caspase-9, caspase-3, Bcl-2, Bax, and β-actin from Santa Cruz Biotechnology and Bid from Abcam Inc. were used according to the manufacturer's instructions.

Cell culture

Human Jurkat T lymphocytes (Jurkat cells) were obtained from the American Type Culture Collection. These cells were maintained in RPMI1640 supplemented with 10 % fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin (100 µg/mL) at sub-confluence in a 95 % air and 5 % CO₂ humidified atmosphere at 37 °C.

Cell viability

Cell viability was evaluated using 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT). Briefly, counted 1 × 10⁶ human Jurkat cells were plated in a 6-well plate and incubated at 37 °C for 24 h. The cells were treated with various concentrations of glycyrol, parthenolide (100 µM, purity ≥ 90 %), or vehicle, and incubated at 37 °C for an additional 24 h. After incubation, cells were collected and co-incubated in MTT assay buffer (0.5 mg/mL MTT in phosphate-buffered saline, PBS) for 2 h, followed by sonication and overnight incubation in a 37 °C CO₂ incubator. Viability was determined using a microplate reader at 595 nm (Emax; Molecular Devices).

DNA fragmentation analysis

The 1 × 10⁶ cells were seeded in a 6-well plate and treated with 5, 25, and 50 µM glycyrol for 3, 6, 12, or 24 h. After incubation, the cells were centrifuged, washed once with cold PBS buffer and centrifuged again for collection. To isolate total DNAs, cells were lysed with 200 µL of lysis buffer (50 mM Tris-Cl, pH 8.0, 200 mM EDTA pH 8.0, 5 % Triton X 100) while incubating on ice for 30 min. After collecting cells by centrifugation at 19 000 g for 10 min, the supernatants were transferred into new 1.5 mL tubes and extracted with 25 : 24 : 1 (v/v) phenol : chloroform : isoamylalcohol. After removing the upper aqueous layer to new 1.5 mL tubes, 1 : 2 (v/v) of 10 M ammonium acetate was added, followed by 1 : 2.5 (v/v) of cold ethanol and kept in a − 20 °C freezer for 2 hours. The DNA pellets were centrifuged, washed once with 75 % ethanol, briefly dried, re-suspended in 30 µL of de-ionized water-RNase solution (0.25 µg/mL RNase in de-ionized water) and incubated at 37 °C for 1 hour. DNA samples were separated in a 2 % agarose gel containing ethidium bromide at 50 V until the loading dye reached two-thirds of the way down the gel, and the resulting DNA bands were visualized and photographed using Doc-It LS Image Analysis software (UVP Inc.).

Flow cytometric analysis

For fluorescent activated cell sorting (FACS) analysis, 2 × 10⁶ cells were directly incubated with 5, 25, or 50 µM glycyrol or vehicle for 6, 12, 16, or 24 h. Cells were stained with Annexin V-FITC and PI according to the manufacturer's directions (BD Pharmingen). Briefly, cells were washed twice with PBS, re-suspended in binding buffer (10 mM HEPES/NaOH [pH 7.4], 140 mM NaCl, 2.5 mM CaCl₂), stained first with FITC Annexin V, and then stained with propidium iodide (PI) before flow cytometry. For cell cycle analysis, harvested cells were washed twice in 1 mL PBS, fixed three times with the reaction volume of absolute ethanol (v/v) at 4 °C for 1 h, centrifuged and washed twice with PBS, stained with 1 mL of PI staining buffer (3.8 mM sodium citrate, 50 µg/mL PI) and treated with 50 µL of RNase A (10 µg/mL), followed by a 3-h incubation at 4 °C. The samples were analyzed using a fluorescent activated cell sorting 440 Flow Cytometer (Becton Dickinson).

Caspase-3 activity assay

Caspase-3 cleavage activity was determined using the fluorogenic substrate Asp-Glu-Val-Asp-(7-amino-4-methylcoumarine) (DEVD‐AMC). Briefly, 2 × 10⁶ cells were incubated for 6 h with different concentrations of glycyrol and harvested and lysed in a lysis buffer containing 25 mM HEPES KOH, 2 mM MgCl₂ (pH 7.4), 10 mM DTT, 2 mM EGTA, 100 µM PMSF, 10 µg/mL leupeptin, 400 ng/mL pepstatin, and 10 µg/mL aprotinin. The lysate samples were frozen and thawed four times using liquid nitrogen (− 196 °C) and a water bath (40 °C) with vortexing and brief centrifugation between steps. Lysates were then centrifuged at 19 000 g for 10 min at 4 °C and the supernatants were collected. Cell lysates (10 µL) were incubated in a 96-well plate with 90 µL of assay buffer (100 mM HEPES‐KOH [pH 7.5], 10 mM DTT) containing 30 µM DEVD‐AMC (Calbiochem). The emitted fluorescence of AMC was measured every minute for 40 min at room temperature at an excitation wavelength of 360 nm and an emission wavelength of 480 nm. Acetyl AMC was used to obtain a standard curve and caspase-3 cleavage activity was calculated in pmol/mg/min. The values were converted to a relative fluorescence unit (RFU) appropriate to a vehicle control value of 1. Actinomycin-D (333 nM, purity by HPLC ≥ 95 %) was used as a positive control.

Fluorescence microscopy

Cellular DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) and examined under fluorescence microscopy. Apoptotic cells were noted by changes in their nuclear morphology, including shrinkage, condensation and margination, and fragmentation of chromatin. In brief, 1 × 10⁶ human Jurkat cells were grown in a 6-well plate, and the sub-confluent cells were treated with 5, 25, and 50 µM glycyrol or vehicle. After a 16-h incubation with the three different concentrations, the cells were collected and washed once with PBS, fixed in 4 % paraformaldehyde in PBS for 30 min, washed 2 times with cold PBS, stained with 2 µg/mL of DAPI in PBS and finally washed 3 times with PBS before fluorescence microscopy (Olympus).

Western immunoblot analysis

Human Jurkat cells (1 × 10⁶) were incubated with 5, 25, or 50 µM glycyrol or vehicle for 0.5, 1, 3, 6, 12, and 24 h. Twenty micrograms of total protein extracts were separated on a 10 % SDS-PAGE gel and electro-transferred to nitrocellulose membranes (Whatman GmbH), blotted to each antibody of anti-Fas, anti-Fas-L, anti-p53, anti-Bcl-2, anti-Bax, anti-caspase-8, anti-caspase-9, anti-caspase-3, anti-Bid, and anti-β-actin and visualized using WEST-SAVE Up™ luminol-based ECL reagent (LabFrontier). The target proteins were detected and photographed using LAS 1000 (Fuji Film) and quantified employing UN-SCAN‐IT™ (Silk Scientific Corp.). N-Acetyl-Glu-Thr-Asp-aldehyde (Ac-IETD‐CHO, purity 99 %, peptide content 91 %) as caspase-3/-8 antagonist and 5-fluorouracil (5-FU, purity by TLC ≥ 99 %) as a positive control were used.

Reverse transcriptase PCR (RT‐PCR)

Jurkat calls (2 × 10⁶) were incubated with 50 µM glycyrol or vehicle for 3 and 6 h. The primer sequences used for PCR amplification were as follows. Fas sense 5′-CACTATTGCTGGAGTCATG‐3′, Fas antisense 5′-CTGAGTCACTAGTAATGTCC‐3′, GAPDH sense 5′-GGTCGGAGTCAACGGATTTGGTCG‐3′, and GAPDH antisense 5′-CTTCCGACGCCTGCTTCACCAC‐3′, respectively. RT‐PCR conditions were the same as previously described [11].

Statistics

Unless otherwise stated, all data were expressed as mean ± standard deviation (SD) from at least three different experiments. A one-way analysis of variance (ANOVA) followed by Dunnett's t-test was applied to assess the statistical significance of the differences among the study groups (SPSS version 10.0). A value of p < 0.05 was chosen as the criterion of statistical significance.

Supporting information

The analysis of apoptosis in Jurkat cells by fluorescence microscopy is available as Supporting Information.

Results and Discussion

Glycyrol was shown to induce dose-dependent cell death in human Jurkat cells with an EC₅₀ of 19.3 µM using the MTT assay and parthenolide as a positive control ([Fig. 1 B]). To determine whether this cell death is apoptotic, Jurkat cells were incubated with different concentrations of glycyrol for 24 h or incubated with a fixed dose (50 µM) for different time durations. Glycyrol induced DNA fragmentation in a dose- and time-dependent manner; we observed that cells treated with 5, 25, and 50 µM glycyrol for 24 h or with 50 µM glycyrol for 3, 6, 12, and 24 h form increasingly fragmented DNAs ([Fig. 1 C]). Endonucleolytic cleavage of genomic DNA into oligonucleosomal fragments along with chromatin condensation is indicative of apoptosis.

In addition, the apoptosis-inducing effect of glycyrol was confirmed using FACS analysis and cell cycle analysis. Glycyrol at 50 µM increased the percentage of apoptotic cells in a time-dependent manner, with apoptotic cells being 37.6 % at 6 h, 52.3 % at 12 h, and 57.7 % at 16 h of incubation. At 20 h of incubation, glycyrol induced apoptosis in a dose-dependent manner: 11.6 % at 5 µM, 22.8 % at 25 µM, and 55.6 % at 50 µM ([Fig. 2 A]). Next, analysis of cellular DNA content by flow cytometry revealed that glycyrol treatments influence cell cycle in Jurkat cells at the S phase, increasing the total cell proportion assigned to S phase from 20.2 % at the control, to 27.4 % at 6 h, 32.8 % at 12 h, 38.9 % at 16 h, and 40.4 % at 20 h ([Fig. 2 B]). Further detailed data should be undertaken to elucidate in more detail how glycyrol influences the cell cycle resulting in apoptosis.

Glycyrol at 5, 25, and 50 µM incubated for 16 h, followed by DAPI staining and fluorescence microscopy, showed the characteristics of apoptotic cell bodies that were clearly observed and whose number increased accordingly in a dose-dependent manner (Supporting Information Fig. 1S). In addition, glycyrol dose-dependently activated caspase-3 shown in the DEVD‐AMC cleavage assay. When converted into relative values referred to untreated control, glycyrol showed relative fluorescence units (RFU) of 2.7 at 5 µM, 3.4 at 25 µM, and 4.8 at 50 µM. The positive control, actinomycin D, exhibited potent DEVD‐AMC cleaving activity of 9.43 (RFU) at 333 nM ([Fig. 3 E]).

Western analysis using various protein antibodies indicated that glycyrol induces the Fas/FasL-FADD‐caspase-8 pathway ([Fig. 3 A]). Fas mRNA and protein levels were increased according to the concentration of glycyrol ([Fig. 3 A] and [D]). Glycyrol induced cleavage of procaspase-8, -9 and -3 and subsequently induced active caspase-3 in dose-dependent manners ([Fig. 3 A]). Further, glycyrol induced caspase-8 cleavage-mediated activation of pro-apoptotic BH3-only protein Bid, which has a unique role as both a Bcl-2 homologue and a BH-3 only protein and links intrinsic and extrinsic apoptosis pathways, indicating that glycyrol can potentiate both a death-receptor pathway and a mitochondrial pathway ([Fig. 3 A]). In parallel, caspase-3/-8 inhibitor, Ac-IETD‐CHO, inhibited cleavage of procaspase-8 and -3 by glycyrol, conversely confirming caspase-8/-3 activating capacity of glycyrol ([Fig. 3 C]).

The positive control 5-FU also induced the cleavage of procaspase-9 and -3, but cleavage of procaspase-8 was not pronounced and further active caspase-3 was not detected. Despite the fact that 5-FU can inhibit DNA synthesis primarily by interfering with the biosynthesis of thymidylate and induce apoptosis in diverse cancer cells [13], it has not much effect on Jurkat cells, reflecting its low cytotoxicity (data not shown).

The tumor suppressor p53 is a potent inhibitor of cell growth that can also induce apoptosis [14]. Glycyrol, however, showed no increasing effects, but a slight lowering effect on p53 protein expression in a dose- and time-dependent manner in human Jurkat cells ([Fig. 3 B]). These results suggest that apoptosis by glycyrol proceeds through a membrane death receptor pathway in human Jurkat cells, independent of p53.

Despite the known NF-κB inhibiting effect of glycyrol [11], protein expression of pro-survival Bcl-2 and proapoptotic Bax (data not shown) were not significantly altered. Further studies are needed to show whether expression of other NF-κB-regulated pro-apoptotic proteins is not also reduced by glycyrol. Protein expression of Bcl-2 and Bax is further known to be regulated by p53 both in vitro and in vivo, and Bax is a direct target of p53 transcriptional activation [15]. Nevertheless, neither Bcl-2 nor Bax is influenced by glycyrol, and even a slight decrease in p53 protein level seems to have no effect on apoptosis by glycyrol in Jurkat cells.

The fact that the protein expression level of the membrane death receptor Fas was increased by glycyrol independently from p53 shows that this natural product acts in a similar manner as many cytostatic active compounds. One such example is curcumin from the rhizome of the Curcuma longa. Curcumin induces a p53 independent apoptosis in human colon cancer and ovarian carcinoma cells [16], [17]. Isoflavone genistein from soybeans also induces apoptosis in lung cancer through a p53 independent pathway [18]. These natural products, including glycyrol, may act as an anti-cancer agent regardless of the status of p53 in cancer cells. Glycyrol may be interesting in such a way that this natural product can be used in combined therapy with other anti-tumor drugs as a sensitizing tool to enhance apoptosis. Before final conclusions are justified, this effect has to be shown in other cancer cell lines and in in vivo experiments.

Acknowledgements

This work was supported by a grant from Korea Food and Drug Administration (2007–2008) for Studies on the Identification of the Efficacy of Biologically Active Components from Oriental Herbal Medicines and was also supported in part by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MEST; No. 20090083533).

References

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Prof. Dr. Yeong Shik Kim

Natural Products Research Institute
College of Pharmacy
Seoul National University

599 Gwanangno Gwanak-gu

Seoul 151-742

Republic of Korea

Phone: + 82 28 80 24 79

Fax: + 82 27 65 47 68

Email: kims@snu.ac.kr

References

• 1 Jacobson M D, Weil M, Raff M C. Programmed cell death in animal development.  Cell. 1997;  88 347-354
  CrossrefPubMedSearch in Google Scholar
• 2 Danial N N, Korsmeyer S J. Cell death: critical control points.  Cell. 2004;  116 205-219
  CrossrefPubMedSearch in Google Scholar
• 3 Hanahan D, Weinberg R A. The hallmarks of cancer.  Cell. 2000;  100 57-70
  CrossrefPubMedSearch in Google Scholar
• 4 Yuan J, Yankner B A. Apoptosis in the nervous system.  Nature. 2000;  407 802-809
  CrossrefPubMedSearch in Google Scholar
• 5 Elmore S. Apoptosis: A review of programmed cell death.  Toxicol Pathol. 2007;  35 495-516
  CrossrefPubMedSearch in Google Scholar
• 6 Nagata S. Fas ligand-induced apoptosis.  Annu Rev Genet. 1999;  33 29-55
  CrossrefPubMedSearch in Google Scholar
• 7 Peter M E, Krammer P H. The CD95(APO-1/Fas) DISC and beyond.  Cell Death Differ. 2003;  10 26-35
  CrossrefPubMedSearch in Google Scholar
• 8 Reidl S J, Shi Y. Molecular mechanisms of caspase regulation during apoptosis.  Nat Rev Mol Cell Biol. 2004;  5 897-907
  CrossrefPubMedSearch in Google Scholar
• 9 Youle R J, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death.  Nat Rev Mol Cell Biol. 2008;  9 47-59
  CrossrefPubMedSearch in Google Scholar
• 10 Tanaka Y, Kikuzaki H, Fukuda S, Nakatani N. Antibacterial compounds of licorice against upper airway tract pathogens.  J Nutr Sci Vitaminol (Tokyo). 2001;  47 270-273
  PubMedSearch in Google Scholar
• 11 Shin E M, Zhou H Y, Guo L Y, Kim J A, Lee S H, Merfort I, Kang S S, Kim H S, Kim S H, Kim Y S. Anti-inflammatory effects of glycyrol isolated from Glycyrrhiza uralensis in LPS-stimulated RAW264.7 macrophages.  Int Immunopharmacol. 2008;  8 1524-1532
  CrossrefPubMedSearch in Google Scholar
• 12 Ying L J, Kim S H, Kim Y S, Kim S A, Kim H S. The first total synthesis of glycyrol.  Tetrahedron Lett. 2008;  49 6835-6837
  CrossrefPubMedSearch in Google Scholar
• 13 Yang L, Wu D, Luo K, Wu S, Wu P. Andrographolide enhances 5-fluorouracil-induced apoptosis via caspase-8-dependent mitochondrial pathway involving p53 participation in hepatocellular carcinoma (SMMC-7721) cells.  Cancer Lett. 2009;  276 180-188
  CrossrefPubMedSearch in Google Scholar
• 14 Vogelstein B, Lane D, Levine A J. Surfing the p53 network.  Nature. 2000;  405 307-310
  PubMedSearch in Google Scholar
• 15 Miyashita T, Reed J C. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene.  Cell. 1995;  80 293-299
  CrossrefPubMedSearch in Google Scholar
• 16 Watson J L, Hill R, Yaffe P B, Greenshields A, Walsh M, Lee P W, Giacomantonio C A, Hoskin D W. Curcumin causes superoxide anion production and p53-independent apoptosis in human colon cancer cells.  Cancer Lett. 2010;  DOI: 10.1016/j.canlet.2010.04.018
  PubMedSearch in Google Scholar
• 17 Watson J L, Greenshields A, Hill R, Hilchie A, Lee P W, Giacomantonio C A, Hoskin D W. Curcumin-induced apoptosis in ovarian carcinoma cells is p53-independent and involves p38 mitogen-activated protein kinase activation and downregulation of Bcl-2 and surviving expression and Akt signaling.  Mol Carcinogen. 2010;  49 13-24
  PubMedSearch in Google Scholar
• 18 Lian F, Li Y, Bhuiyan M, Sarkar F H. p53-independent apoptosis induced by genistein in lung cancer cells.  Nutr Cancer. 1999;  33 125-131
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Yeong Shik Kim

Natural Products Research Institute
College of Pharmacy
Seoul National University

599 Gwanangno Gwanak-gu

Seoul 151-742

Republic of Korea

Phone: + 82 28 80 24 79

Fax: + 82 27 65 47 68

Email: kims@snu.ac.kr

• Supplementary Material