Journal of Cancer Prevention 2013; 18(2): 169-176
Published online June 30, 2013
© Korean Society of Cancer Prevention
Seaho Kim1, Dong Hwan Kim1, Sun Hwa Lee1, Min Jeong Kim1, Jeong-Hyun Yoon1, Hae Young Chung1, Chun Soo Na2, and Nam Deuk Kim1
1Department of Pharmacy, College of Pharmacy, Molecular Inflammation Research Center for Aging Intervention (MRCA), Pusan National University, Busan, 2Lifetree Biotech Co., Ltd., Suwon, Korea
Correspondence to :
Nam Deuk Kim, Department of Pharmacy, Pusan National University, 63 Beon-gil 2, Busandaehag-ro, Geumjeong-gu, Busan 609-735, Korea Tel: +82-51-510-2801, Fax: +82-51-513-6754, E-mail: email@example.com
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Urushiols are mixtures of olefinic catechols which is isolated from the sap of Korean lacquer tree (
The cytotoxicity of urushiols was assessed by MTT assays on the two gastric adenocarcinoma cell lines, MKN-45 (wild type of p53) and MKN-28 (mutant type of p53). We also examined the action mechanisms of urushiol by analyzing its effects on cell cycle progression and apoptosis induction.
The cytotoxic results from MTT assays indicated that urushiol inhibited human gastric cancer cell growth in a dose-dependent manner, with IC50 values of approximately 15 and 20
These data provide evidence that urushiol has the potential to be used as a chemotherapeutic agent in human gastric cancer.
Keywords: Urushiol, Gastric cancer cells, Apoptosis, G1 arrest
Urushiols are mixtures of olefinic catechols with n-C15 or n-C17 alkyl side chain which is isolated from the sap of Korean lacquer tree (
Gastric cancer is the most prevalent cancer in the world, including Korea and Japan. Despite recent advances in cancer treatments, most newly diagnosed gastric cancer patients are incurable and have a median survival of less than one year.9 It is well known that gastric cancer can be divided into two major categories: the intestinal, expanding, or differentiated type; and the diffuse, infiltrative, or undifferentiated type.10 The differentiated type is characterized by expansive growth and liver metastasis; whereas the undifferentiated type is distinguished by infiltrative growth and peritoneal dissemination.11 MKN-45 and MKN-28 cell lines were derived from human gastric carcinoma.12 The MKN-45 cell line was derived from a metastatic liver tumor of a 62-year-old female with gastric cancer. The parent tumor of MKN-45 cells was a poorly differentiated adenocarcinoma of the medullary type. The MKN-28 cell line was derived from a metastatic tumor in a cervical lymph node of a 70-year-old female with gastric cancer. The parent tumor of MKN-28 cells was a moderately differentiated tubular adenocarcinoma. MKN-45 cell line contains wild type of p53 genes, whereas MKN-28 cell line has mutant type of p53 genes.13 In this study, we examined whether urushiol inhibits cell growth and promotes cell death. Furthermore, we studied potential molecular mechanisms of action by which urushiol inhibit cancer cells if it did.
MKN-45 and MKN-28, human gastric cancer cell lines, were obtained from Korean Cell Line Bank (Seoul, Korea). All cells were grown in RPMI 1640 (Gibco BRL, Grand Island, NY, USA) with 10% fetal bovine serum in a humidified 5% CO2 at 37°C containing 50 mg/ml gentamicin, and 135 mg/ml glutamine. The medium was changed three times per week. The number of cells was counted with a hematocytometer. Urushiol complex was obtained in a solution form from Lifetree Biotech Co., Ltd. (Suwon, Korea). The aqueous solution was filtered through Whatman No. 2 filter paper, and then concentrated in vacuum to dryness. The yield of urushiol was about 3.5%. The powder was weighted and adjusted to a final concentration with DMSO (Sigma-Aldrich, St. Louis, MO, USA) and stored at ?20°C until use. The stock solution of urushiol was diluted with medium to the desired concentration prior to use. The maximal concentration of DMSO did not exceed 0.1% (v/v) in the treatment range (2?10
Assessment of cell proliferation by MTT assay
The proliferation of gastric cancer cells was assessed by using the MTT assay which is based on the conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to MTT-formazan by mitochondrial enzyme.14 In brief, these cells were cultured with 1 ml of medium per well in 24-multiwell plates at 37°C for 24 h under 5% CO2 in air. The cells were then treated with desired concentrations of urushiol. Then, the MTT solution (5 mg/mlPBS) was added to the well with a concentration of 0.25 mg/ml. Following the incubation for 4 h, the mixture of medium and the MTT solution was carefully discarded, after which 1 ml EtOH-DMSO (1 : 1) was added to the well in order to extract the crystallized dye. The amount of blue dye formed was determined by measuring the absorbance at 540 nm. Measurements were performed in triplicates.
Observation of nuclear structure
After the cells were treated with or without half-maximal inhibition (IC50) doses of urushiol (15
Agarose gel analysis of DNA fragmentation
Cells were harvested, rinsed twice in cold PBS, and resuspended in lysis buffer [5 mM Tris-HCl (pH 7.5), 5 mM ethylenediaminetetra acetic acid (EDTA), and 0.5% Triton X-100] at 4°C for 30 min. After centrifugating them at 27,000×g for 15 min, the supernatant was treated with RNase, followed by proteinase K digestion, phenol/chloroform/isoamyl alcohol (25 : 24 : 1) extraction and isopropanol precipitation. DNA separated through a 1.5% agarose gel was stained with ethidium bromide (EtBr, Sigma-Aldrich) and visualized by ultraviolet light source.
Morphological study of apoptotic cells
After the cells were treated with or without IC50 doses of urushiol (15
Gel electrophoresis and Western blotting
The cells were harvested, lysed, and protein concentrations were quantified using the Bio-Rad protein assay (Bio-Rad Lab., Hercules, CA, USA) by the procedure described by the manufacturer. For the Western blot analysis, an equal amount of protein was subjected to electrophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA) by electroblotting. Blots were probed with the desired antibodies for 1 h, incubated with diluted enzyme-linked secondary antibodies and then visualized by the enhanced chemiluminescence (ECL) according to the recommended procedure (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). The primary antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and Calbiochem (Cambridge, MA, USA). Peroxidase-labeled secondary antibodies were purchased from Amersham Pharmacia Biotech.
Data were expressed as the mean±SD of three separate experiments and analyzed by Student’s
The effect of urushiol on the viability of MKN-28 and MKN-45 cells was assessed via MTT dye assay at the concentrations of 3, 6, 12, and 24
There were significant morphological changes on MKN-45 and MKN-28 cells after the urushiol treatment (Fig. 2). However, the morphologies of two cell lines were significantly different. The sizes of MKN-45 cells were smaller than MKN-28 cells after urushiol treatment. The shape of MKN-45 cell was round, whereas MKN-28 cell was polygonal. Although apparent morphological changes were observed on urushiol-treated MKN-45 (Fig. 2A and B) and MKN-28 cells (Fig. 2C and D), any other differences between two cells were not detectable by light microscopy with no staining. To test whether decreased viability of human gastric cancer cells by urushiol was attributed to apoptosis, the structures of nucleus were studied with PI staining on urushiol-treated cells. In MKN-45 cells, morphological changes were corresponding to apoptosis, including nuclear fragmentation and condensation, observed by the treatment of 15
Urushiol induced apoptosis in MKN-45 cells
To identify a hallmark of apoptotic cell death, assay for the genomic DNA fragmentation was performed. Urushiol treatment invoked DNA ladder formation, indicating that urushiol induced apoptosis in MKN-45 cell. The formation of DNA ladder was increased concentration-dependently (Fig. 3A). However, urushiol did not mediate DNA ladder formation on MKN-28 cells (Fig. 3B).
To confirm apoptotic cell death of MKN-45 by urushiol treatment, the cellular morphology was investigated via electron microscopy. The degeneration of apoptotic bodies observed on MKN-45 with the treatment of urushiol (Fig. 4A and B), whereas MKN-28 cells maintained intact cellular structure (Fig. 4C and D).
The p53 tumor suppressor gene is a cell cycle regulator and is able to induce cell cycle arrest to allow DNA repair or apoptosis.16 The product of
Caspase-3 has known as executioner of apoptosis that mediated DNA fragmentation and cleavage of many cellular and nuclear proteins.20 To study the involvement of caspase-3, the activation of caspase-3 and PARP cleavage were observed on MKN-45 cells treated with urushiol. After treatment of urushiol the level of proenzyme form (32 kDa) of caspase-3 was increased and active form (19 kDa) of enzyme was also detected on MKN-45 cells (Fig. 5). poly(ADP-ribose) polymerase (PARP), one of well-known substrates of caspase-3, was cleaved on MKN-45 cell by urushiol treatment (Fig. 5).
Urushiol mediated G1 arrest on MKN-28 cells
Although urushiol mediated apoptosis-associated changes on MKN-45 cell, it seemed that urushiol did not induce apoptosis on MKN-28 cells. DNA ladder formation and nuclear condensation were not observed after treatment with urushiol on MKN-28 cell (Fig. 3B, 4D). This implies that other mechanisms of growth inhibitory effect of urushiol on MKN-28 might be involved. To investigate the effects of urushiol on cell cycle, we studied expression of p21
In the present study we observed urushiol inhibited the proliferation of human gastric cancer cell lines (MKN-45 and MKN-28). This is the first study to report cytotoxic effects of urushiol on human gastric cancer cells. Urushiol showed cytotoxic effects on the proliferation of human gastric cancer cell lines. Of note, although urushiol exerted cytotoxic effects on both MKN-45 and MKN-28 cell lines, the involved mechanisms of cell death seems to be different.
It has been reported that enhanced apoptosis is responsible for the effects of chemotherapy and for the regression of tumor.21 Moreover, the relationship between apoptosis and the chemosensitivity of cancer cells has been analyzed extensively. From recent studies, it was suggested that the wild-type p53 protein could channel DNA-damaged cells toward apoptosis, while the product of the
The binding of agonistic anti-Fas antibody of Fas ligand to Fas receptor result in transduction of a cytolytic signal to the cell, followed by apoptosis.18 This was well examined in a variety of leukemia/lymphoma cell lines
The activation of caspase-3 and the cleavage of PARP have used as early markers of apoptosis.20 After treatment of urushiol, activation of caspase-3 and concomitant PARP cleavage were observed in MKN-45 cells (Fig. 5). Fragmentation of cellular DNA at the internucleosomal linker regions has been observed in cells undergoing apoptosis induced by a variety of agents. This cleavage produces ladders of DNA fragments that are the sizes of integer multiples of a nucleosome length (180?200 bp).25 Urushiol induced DNA ladder formation on MKN-45 cells with nuclear condensation (Fig. 3A, 4B). However, it was not observed that the DNA fragmentation (Fig. 3B) and nuclear condensation (Fig. 4D) in MKN-28 cell by urushiol treatment. To investigate the morphological changes induced by urushiol treatment, the total cellular morphology was examined under electron microscopy. Urushiol induced significant morphological changes indicating apoptosis. The chromatin was condensed and became segregated into sharply delineated masses. Moreover, the cytoplasmic condensation and segregation also observed on urushiol-treated MKN-45 cells (Fig. 2F, 4A).
The results presented here demonstrated that the treatment with urushiol on MKN-45 cells induced apoptosis, and this was closely linked with the Fas/Fas ligand signal, p53 induction, decreased Bcl-2/Bax ratio, caspase-3 activation, and DNA ladder formation. The mechanisms of action of urushiol have not been fully known to date. It is possible that urushiol directly binds to Fas receptor, and induces apoptosis through caspase activation. In addition, increased Bax protein mediates caspase activation by urushiol, followed by up-regulation of p53 protein. In contrast to MKN-45 cells showed several apoptosis-associated signals by urushiol treatment, urushiol seemed to mediate cell cycle arrest at G1 phase on MKN-28 cells. CDK inhibitor proteins p21
In conclusion, these data suggest that urushiol induced apoptosis in MKN-45 cells and mediated cell cycle arrest in MKN-28 cells at G1 phase through different pathway. Understanding such mechanisms of cell death and cell cycle arrest may allow us to enhance apoptotic responses in human gastric cancer cells for tumor regression. Thus, urushiol could be used as a candidate for developing chemotherapeutic agent in human gastric cancer cells.
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