Shikonin time-dependently induced necrosis or apoptosis in gastric cancer cells via generation of reactive oxygen species
Mu-Jang Lee a,1, Shao-Hsuan Kao b,1, Jing-En Hunag b, Gwo-Tarng Sheu c, Chi- Wei Yeh b, You-Cheng Hseu d,
Chau-Jong Wangb,e,⇑, Li-Sung Hsu b,e,⇑
a Cardiovascular Center, Antai Tian-Sheng Memorial Hospital, Pingtung 92843, Taiwan
b Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan
c Institute of Medicine, Chung Shan Medical University, Taichung 40201, Taiwan d Department of Cosmeceutics, College of Pharmacy, China Medical University, Taichung 40402, Taiwan
e Clinical Laboratory, Chung Shan Medical University Hospital, Taichung 40201, Taiwan
Keywords: Apoptosis, Gastric cancer, Necrosis, Shikonin a b s t r a c t
The effects of shikonin on gastric cancer cells were investigated in this study.
Exposure to shikonin reduced the viability of gastric cancer cells in a time- and dose- dependent manner. However, apoptosis was not observed in gastric cancer cell treatment with different concentrations of shikonin for 6 h. By contrast, treatment with shikonin for 24 h significantly induced apoptosis, as evidenced by the results of TUNEL assay and flow cytometry analysis in proportion to the concentration.
Disruption of the mitochondrial membrane potential was observed in gastric cancer cells that were treated with shikonin for 6 and 24 h. Pretreatment with necrostatin-1 recovered cell death and mitochondrial membrane potential in the 6 h shikonin treatment, but not in the 24 h shikonin treatment. Western blot results reveal enhanced p38 phosphorylation, downregulated AKT phosphorylation, and increased caspase3 and PARP cleavage in cells that were treated with shikonin for 24 h, but not in cells treated for 6 h. Shikonin also triggered reactive oxygen species (ROS) generation both in the 6 and 24 h treatments. Pretreatment with N-acetylcysteine blocked shikonin- induced cell death. In summary, our findings suggest that shikonin, which may function as a promising agent in the treatment of gastric cancers, sequentially triggered necrosis or apoptosis through ROS generation in gastric cancer cells.
1. Introduction
Shikonin, one of the major bioactive components of Lithospermum erythrorhizon, is widely used in traditional Chinese medicine in treating measles, sore throat, and burns
[1]. Shikonin has been shown to exert multiple biological effects, such as the inhibition of PMA-induced cyclooxygenase 2 expression [2], induction of glucose uptake in 3T3L1 cells and skeletal muscle cells [2,3], and inhibition of
lipopolysaccharide-mediated tumor necrosis factor a release via the proteasome pathway [4]. Recently, shikonin has been extensively studied for its anti-tumor
activities. Shikonin administration inhibits tumor growth and prolongs the life span of sarcoma S180-bearing mice [5]. Treatment with shikonin also activates the caspase pathway, which induces the apoptosis of several human cancer cells, such as leukemia HL-60 cells [6], colorectal cancer cells [7], and osteosarcoma [8]. Shikonin blocks the signal mediated by the epithelial growth factor receptor and decreases extracellular signal-regulated kinase (ERK) activities, which can diminish cell proliferation in human epidermoid carcinoma cells [9]. In hepatoma cells, shikonin triggers apoptosis through increased reactive oxygen species (ROS) production, and subsequently downregulates AKT and RIP1/NFjB activities [10]. Chen et al. showed that shikonin also elevates ROS generation, decreases glutathione concentration, and disrupts the mitochondrial membrane potential, which results in apoptosis in human glioma cells [11]. Han et al. demonstrated that shikonin treatment causes drug-resistant cancer cells to undergo necroptotic death [12]. In addition, these authors also showed that pretreatment with antioxidant agents, such as N-acetylcysteine (NAC), pifithrin-a, or cyclosporin A, abolishes shikonin-induced cell death [11].
Gastric adenocarcinoma is one of the leading causes of cancerrelated deaths worldwide. The recurrent rate and mortality remain
unexpectedly high because of the lack of effective chemotherapeutic regimens. The anti-cancer abilities of traditional Chinese medicine or dietary food extracts with low toxicity and side effects have
gained increasing interest among researchers. For instance, epigallocatechin- 3-gallate, which is a polyphenol extracted from green
tea, displays anti-tumor activities by triggering apoptosis through the downregulation of gastric cancer cell survival [15]. Li et al.
demonstrated that triptolide enhances the sensitivity of gastric cancer cells to cisplatin-induced apoptosis [16]. Previous studies indicated that shikonin inhibits proliferation of human cancer cells, including hepatocellular carcinoma (HCC) [10] and glioma [11], but not normal cells, such as HEK 293 and LO2 cells [10]. However, the effects of shikonin on gastric cancer cells remain unclear. In the present study, we showed that shikonin induces both necrosis and apoptosis of gastric cancer cells in a time-dependent manner.
This study is the first report to demonstrate the molecular mechanisms
of shikonin in gastric cancer cell death.
2. Materials and methods 2.1. Materials
All chemicals were purchased from Baker or Sigma Chemical
(St. Louis, MO, USA). The phospho-p38 antibody was obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against p38, phospho-ERK, total ERK, phospho-AKT, and total AKT were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Anti-b actin and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Sigma Chemical Company. Fetal
bovine serum (FBS) and penicillin–streptomycin mixture were obtained from Gibco Laboratory (Gaithersburg, MO, USA). Shikonin
was purchased from Calbiochem (La Jolla, CA, USA). Before each experiment, shikonin was freshly dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 4 mMand subsequently diluted to final concentration by culture medium. The final DMSO content was 0.1%.
2.2. Cell culture
Human gastric cancer AGS, AZ521, and SCM-1 cell lines were obtained from American Type Culture Collection. These cell lines were maintained in RPMI supplemented with 10% FBS, 1 mM sodium pyruvate, 4 mM L-glutamine, 1.5 g/L sodium bicarbonate,
4.5 g/L glucose, 100 unit/mL penicillin, and 100 lg/mL streptomycin at 37 C in a humidified atmosphere containing 95% air and
5% CO2.
2.3. 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay
Human AGS, AZ521, and SCM-1 cells were seeded in 24-well
plates at a density of 5 104/mL, and treated with indicated concentrations of shikonin for 6 or 24 h. After removing the medium,
the cells were incubated with a medium containing 5.0 g/L MTT at 37 C for 2 h. After washing with PBS, the purple-blue formazan was dissolved in 1 mL of isopropanol, and the absorbance was measured at 563 nm. The relative cell viability percentage was determined by the absorbance of OD563 in shikonin-treated groups compared with the vehicle groups.
2.4. Flow cytometric analysis
AGS cells were treated with indicated concentrations of shikonin
for 24 h. After washing with PBS, the cells were detached by trypsinization and fixed in 70% ethanol overnight at 20 C. The cells were then washed with ice-cold PBS, and reacted with
50 lg/mL propidium iodine (PI) in the dark for 15 min. The cell cycle distribution was measured using BD biosciences FACscan with CellQuestTM Pro software. The percentage of apoptosis was detected by counting the fraction of cells with DNA contents at the
sub-G1 phase.
2.5. Detection of ROS
Intracellular ROS was determined using a 6-corboxy-20,70- dichlorodihydrofluoroscein diacetate (DCFDA) fluorescent probe.
The AGS cells were plated in 6-well plates and allowed to attach overnight. After treatment with the indicated concentrations of shikonin for 24 h, the cells were loaded with 10 lM DCFDA at 37 C for 1 h. Fluorescence was detected using Molecular Devices Flexstation 3.
2.6. Measurement of mitochondrial membrane potential by JC-1 staining
The AGS cells were cultured in 6-well plates, and treated with the indicated concentrations of shikonin for 6 and 24 h. The cells were incubated with 0.15 mM JC-1 in the dark for 15 min and
washed with PBS. The signals were captured by an upright fluorescence microscope (ZEISS AXioskop2) with an emission filter of
515 nm and an excitation filter of 450–490 nm. Red signals indicated that the cells had a higher mitochondrial membrane potential,
whereas green signals showed cells with a lower mitochondrial membrane potential.
2.7. Western blot analysis
The AGS cells were treated with the indicated concentrations of
shikonin for 24 h. The cell lysates were then extracted. Protein concentrations were measured using a Bradford protein assay kit (Bio-
Rad, USA). Approximately 50 lg of protein was separated by 10%
polyacrylamide gel and electrotransferred into a nitrocellulose membrane. The membrane was blocked by PBS containing 0.5%
non-fat milk for 1 h at room temperature. The membrane was then probed with indicated primary antibodies overnight at 4 C. The membrane was washed with PBS containing 0.1% Tween-20
(PBST). The membrane was then reacted with HRP-conjugated bovine
anti-goat IgG antibody (Santa Cruz Biotechnology, USA;
1:5000 dilution) after extensive washing with PBST and detection
of reactive signals using enhanced chemiluminescence (ECL commercial kit, Amersham Pharmacia Biotech, UK). b-actin expression
was used as the loading control value.
2.8. Statistical analysis
Data are reported as mean ± SD of the independent experiments, and were evaluated by Student’s t-test. P < 0.05 was considered to be statistically significant.
