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Full-length article Acta Pharmacologica Sinica 2005 October; 26 (10): 1248-1252 Anticancer activity of sodium caffeate and its mechanism1 Feng XU2,3,4, Sheng-hua ZHANG3, Rong-guang SHAO3, Yong-su ZHEN3 2Department of Pharmacology, Zhujiang Hospital, Southern Medical University, Guangzhou 510280;
3Department of Oncology, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
1Project supported by the National Key Basic Research Development Program of China
(No 2002CB513108).
Aim: To study the anticancer activity of sodium caffeate
(SC). Methods: A nucleoside transport assay was used to analyze
the inhibitory effects of SC on nucleoside rescue. The MTT assay was used to measure cell proliferation. Flow cytometry
was used to measure the apoptosis of BEC-7402 induced by SC and the cell cycle distribution change . Western blotting
analysis was employed to investigate Bcl-2, caspase and Bax expression. Intracellular
Ca2+ and mitochondrial membrane potential were determined by flow cytometry.
In vivo anti-tumor activity was measured using a tumor transplantation model
in mice. Results: SC inhibited the nucleoside transport of BEL-7402 cells with an
IC50 of 1.02 mg/mL. SC inhibited tumor cell
proliferation with an IC50 between 100 µg/mL and 200 µg/mL. SC induced BEL-7402 cell apoptosis in a time- and
dose-dependent manner, which was induced by arresting cells in S phase. The
in vivo study showed that tumor growth was
inhibited in a dose-dependent manner. Activated caspase-3 and Bax expression were up-regulated after treatment with SC,
while Bcl-2 expression was down-regulated. Intracellular
Ca2+ was increased while mitochondrial membrane potential was
decreased by SC. Conclusion: SC is a new anticancer agent with promising potential. Key words caffeic acid; anticancer activity; cell division; cell apoptosis; cell cycle; phytogenic antineoplastic agent; flow cytometry; Western blotting Note: Please read the complete
full text with Figures and Tables at
Introduction
There have been an increasing number of anticancer
phytochemicals identified in our daily diet. Some of the most
promising and extensively investigated are those present in
the cruciferous family of vegetables, alliums and tea.
Phytochemicals should be considered as an inexpensive and
readily applicable, acceptable and accessible approach to
cancer control and management for general populations. This
is particularly important considering the sluggish progress
made in cancer treatment. It is still an urgent task to seek
new anticancer drugs from natural resources in oncology
pharmacology.
Cinnamic acid is one of the phytochemicals with
potential chemopreventive effects in preventing
carcinogenesis[1,2]. Cinnamamide, a natural compound containing the cinnamic
acid structure, is a new antitumor agent that acts on matrix
metalloproteinase, which has been demonstrated by
previous work in our laboratory[3]. Caffeic acid
(3,4-dihydroxycinnamic acid) is a polyphenol that is found in coffee, fruits,
vegetables, grains and many others
plants[4-7]. It is also particularly abundant in propolis beehives with 20%-25%
content and has various pharmacological activities, such as
antioxidant and antiviral effects[8]. The anticancer effect of
caffeic acid, however, has not been reported up to now.
Because caffeic acid is prone to air oxidation and is only
slightly soluble in water, its stable sodium salt (sodium
caffeate, SC) was prepared in our laboratory and used in the
present study. Here we report the antitumor effect of SC
both in vitro and in vivo.
Materials and methods
Reagents RPMI-1640 medium was purchased from
Gibco BRL (Gaithersburg, Maryland, USA ). Fetal calf serum
(FCS) was purchased from Hyclone (Logan, Utah, USA).
MTT, nonidet P-40(NP-40), phenylmethylsulfonyl
fluoride(PMSF), aprotinin, ponceau S, Triton X-100, propidium
iodide, Fluo-3, rhodamine 123, RNase A, proteinase K,
Hoechst 33342 and other reagents were purchased from Sigma
( StLouis , Missouri, USA). [H3]TdR was purchased from
Chinese Atomic Energy Institutes (Beijing, China). Annexin
V-FITC/PI apoptosis detection kit was purchased from
BioVision company (Hannover, Germany). Mouse
anti-Bcl-2 monoclonal antibody, mouse anti-caspase-3 monoclonal
antibody and rabbit anti-bax polyclonal antibody were
products of Calbiochem (San Diego, California, USA).
Horseradish peroxidase-conjugated secondary anti-mouse antibody
and anti-rabbit antibody were products of Santa
Cruz Biotechnology, Inc(Santa Cruz, California,USA). Enhanced
luminol reagent and oxidizing reagent were products of NEN
Life Science Products (Boston, Massachusetts, USA). Dr
Dan-qing SONG in Department of Chemistry, Institute of
Medicinal Biotechnology (Beijing, China) synthesized the SC.
Cells and carcinoma Human oral cavity epidermis
squamocellular carcinoma cell line (KB), human hepatocarcinoma cell line (BEL-7402) and human acute
promyelocytic cell line (HL-60) were purchased from the
Shanghai Institute of Cell Biology, Chinese Academy of
Sciences (Shanghai, China), and were grown routinely in
RPMI-1640 supplemented with 10% heat-inactivated FCS. The
medium was supplemented with 100 U/mL penicillin, 100 µg/mL
streptomycin and 2 mmol/L glutamine, and the cells were
incubated in a humidified atmosphere, with 5%
CO2 in air at 37 °C.
Mouse-transplanted hepatocarcinoma H22 and mouse
colorectal cancer C26 cell lines were maintained by serial
transplantation into mice in our laboratory. Kunming
species mice were supplied by the Experimental Animal Center,
Chinese Academy of Medical Sciences (Beijing, China).
Nucleoside transport assay[9] Briefly, cells in the
logarithmic growth phase were harvested. The cell suspension
was prepared with RPMI-1640 medium at
5×106 in 0.9 mL in each test tube. Different concentrations of SC in 0.1 mL
RPMI-1640 were added and the tube was kept in a water bath
at 37 °C for 5 min. Phosphate-buffered solution (PBS) was
used as a control. RPMI-1640 0.1 mL (containing
3.7×104 Bq of [3H]TdR in medium free of serum) was added for 30 s and
5 mL ice-cold normal saline was added to terminate the
reaction. The reaction was filtrated through a GF/B glass
fiber filter (Whatman International, Maidstone, UK) under
vacuum. The filters were washed with 0.2 mL of 1 mol/L
NaOH and 0.5 mL ethanol, dried under vacuum and placed
in scintillation vials containing 2 mL of dimethylbenzene with
0.4% PPO/0.01% POPOP. The cpm (counts per minute) were
measured using an LS-9800 scintillometer (Beckman Instruments, Fullerton, California, USA).
MTT assay Briefly, cells in the logarithmic growth phase
were harvested and seeded in 96-well plates (Costar,
Cambridge, Massachusetts, USA) overnight. The test
compound was added and cells were further incubated for 72 h.
The viability of cells was determined using the MTT assay
according to the method described by Carmichael
et al [10].
Long-term clonogenicity Cell survival was tested using
a clonogenic assay, as described by Valduga et
al[11]. Briefly, cells in the logarithmic growth phase were harvested
and 250 cells/mL of a single-cell suspension was prepared
with medium. The cell suspension 200 µL was seeded in
96-well plates (50 cells/well) overnight, and the test compound
was added. After 1 week of incubation at 37 °C in air with 5%
CO2, colonies were counted.
Flow cytometry After appropriate treatment, cells were
harvested by centrifugation and washed with PBS. The cells
were fixed with ice-cold 75% ethanol for 18 h at 4 °C. The
cell apoptosis was measured according to the protocol of
Annexin V-FITC/PI apoptosis detection kit. Cell cycles
change was measured by treatment of the fixed cell
suspensions which were washed with PBS and stained with 80 µL of
50 µg/mL propidium iodide and 50 µg/mL RNase A for 30 min
in the dark. Samples were run through an EPICS XL flow
cytometer (Coulter, Miami, Florida, USA). Results are
presented as the number of cells versus the amount of DNA as
indicated by the intensity of
fluorescence[12]. The results on flow cytometry represented the average of 3 individual
experiments.
Western blotting assay The cells were lysed in lysis
buffer at 4 °C with sonication. The lysates were centrifuged
at 15 000×g for 15 min and the concentration of protein in
each lysate was determined using Coomassie Brilliant Blue
G-250. Loading buffer was added to each lysate, which was
subsequently boiled for 3 min and then electrophoresed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Proteins were transferred to nitrocellulose and incubated with
anti-Bcl-2, anti-caspase-3 or anti-Bax antibodies and then
with peroxidase-conjugated secondary antibodies.
Detection was carried out using an enhanced chemiluminescence
agent[13,14]. The results on Western blot analysis represented
the average of 3 individual experiments.
Intracellular Ca2+ and mitochondrial membrane
potential (DY) Following appropriate treatment, cells were
collected by centrifugation and incubated at 37 °C with
Fluo-3 for 40 min and then with rhodamine 123 for 20 min. Cells
were then washed 3 times with cold PBS, and the
intracellular Ca2+ concentration and Dy were measured by flow
cytometry[15-17].
Tumor transplantation and drug administration
H22 ascites were diluted to
7.5×105/mL suspension and 0.2 mL of
the cell suspension was inoculated subcutaneously into the
right axilla of each mouse of the Kunming species (weighing
20±2 g). A C26 tumor-cell suspension was prepared by
gently suspending tumor tissue in normal saline (1 g of tumor
tissue with 3 mL of normal saline) in a cold water bath and
inoculated as above. After 24 h of tumor cell inoculation, SC
was administered intraperitoneally for 10 d. Normal saline
was used as a control. On the d 11 the mice were killed, and
body weight and tumor tissue were weighed.
Statistical analysis The data are the mean values of at
least 3 experiments and are expressed as mean±SD. The
Student’s t-test was used to compare data.
P<0.05 was considered to be statistically
significant.
Results
Inhibition of nucleoside transport by SC SC inhibited
nucleoside transport in the hepatocarcinoma BEL-7402 cell
line with an IC50 of 1.02 mg/mL. However, the inhibiting
effect on nucleoside transport was not very strong.
Inhibition of proliferation and induction of apoptosis by
SC SC inhibited tumor-cell proliferation with an
IC50 of between 100 µg/mL and 200 µg/mL (Table 1). Inhibition of
BEL-7402 cells proliferaction by SC was dose-dependent and
time-dependent (Figure 1).
Flow cytometry showed that SC induced BEL-7402 cell
apoptosis in a time- and dose-dependent manner (Table 2).
After 24 h of treatment with SC, the cell cycle changed. The
percentages of cells in S phase increased markedly while
percentages of cells in G2/M phase decreased, which
suggested the apoptosis was induced by arresting the cells in S
phase (Table 3).
Inhibition of clonogenicity by SC SC inhibited cell
clonogenicity with an IC50 between 0.5 µg/mL and
3 µg/mL (Table 4). SC was capable of inhibiting the different cell lines
to different extents.
Effect of SC on transplanted tumor growth
H22 and C26 were inoculated subcutaneously into mice. After SC
administration for 10 d, tumor growth was inhibited in a
dose-dependent manner. No significant difference in body weight
was found between the groups, suggesting that SC does
not show toxicity in vivo (Table 5 and 6).
Effect of SC on expression of apoptosis-associated
proteins The bands were scanned with light density. Activated
caspase-3 and Bax expression were up-regulated after SC
treatment, while Bcl-2 expression was down-regulated
(Figure 2).
Effect of SC on intracellular Ca2+
and mitochondrial membrane potential After treatment with
10 mg/mL SC for 24 h, intracellular
Ca2+ was increased 1.78-fold compared to
the control. Dy was decreased by 22.7% compared to the
control. The results showed that SC increased intracellular
Ca2+ levels and decreased Dy.
Discussion
Most anti-metabolites in tumor chemotherapy inhibit
nucleoside de novo synthesis but can not block nucleoside
rescue in cancer cells. It is therefore important to control
nucleoside rescue by inhibiting nucleoside transport. Previous work found that dipyridamole enhanced the anticancer
effect of acivicin by inhibiting nucleoside
transport[9].The present study demonstrated that SC was a new member of
the nucleoside transport inhibitor family.
Caffeic acid is an active phytophenol that has been found
to inhibit rat glutathione-S-transferase isoenzymes both
in vitro and
in vivo[18]. A large number of population-based
studies have found that consumption of wholegrains,
vegetables and fruits abundant in caffeic acid reduces the risk of
cancer[19-21]. The aqueous extract of
Salvia miltiorrhiza, a traditional Chinese herb containing caffeic acid was found
to strongly inhibit the proliferation of human hepatoma
HepG2 cells. It was also observed that its crude extract caused
apoptotic cell death[22]. Salvianolic acid A, a caffeic acid
trimer, showed synergistic effects in combination with other
antitumor agents. Further, salvianolic acid A could increase
the antitumor effects of 5-flurouracil without increasing its
toxicity in an animal study [2]. However, no report has been
published on the anticancer effects of caffeic acid either
in vitro or in vivo. We are the first to report that the sodium
salt of caffeic acid inhibits proliferation of cancer cells, with
IC50 between 100 µg/mL and 200 µg/mL. Further, we showed
that it induced the apoptotic cell death and changed
cell-cycle distribution by arresting cells in S phase. The
in vivo study showed that SC inhibited the tumor growth of
transplanted H22 and C26 cells in mice with an inhibition rate of
42%-54% when treated with 1 g/kg SC for 10 d.
Preliminary studies on the anticancer mechanism of SC
demonstrated that after treatment with SC, Bcl-2 expression
was down-regulated and mitochondrial membrane
permeabi-lity was changed. The mitochondrial permeability transition
pore was opened and the mitochondrial membrane
potential was broken up. The mitochondrion was swelled and in
a state of hyperosmosis before apoptosis was induced.
Meanwhile cytochrome c was released, caspase-3 was
activated in the presence of Apaf-1 and caspase-9, and apoptosis was
induced[23-27].
References
1 Liu L, Hudgins WR, Shack S, Yin MQ, Samid D. Cinnamic acid:
a natural product with potential use in cancer intervention. Int
J Cancer 1995; 62: 245-50.
2 Jiang RW, Lau KM, Hon PM, Mak TC, Woo KS, Fung KP.
Chemistry and biological activities of caffeic acid derivatives
from Salvia miltiorrhiza. Curr Med Chem 2005; 12: 237-46.
3 Jiang XF, Zhen YS. Cinnamamide, an antitumor agent with low
cytotoxicity acting on matrix metalloproteinase. Anticancer
Drugs 2000; 11: 49-54.
4 Radtke J, Linseisen J, Wolfram G. Phenolic acid intake of adults 5 Eberhardt MV, Lee CY, Liu RH. Antioxidant activity of fresh
apples. Nature 2000; 405: 903-4.
6 Mattila P, Kumpulainen J. Determination of free and total
phenolic acids in plant-derived foods by HPLC with diode-array
detection. J Agric Food Chem 2002; 50: 3660-7.
7 Bryngelsson S, Dimberg LH, Kamal-Eldin A. Effects of
commercial processing on levels of antioxidants in oats
(Avena sativa L). J Agric Food Chem 2002; 50: 1890-6.
8 Greenaway W, Scaysbrook T, Whatley FR. The analysis of bud
exudate of Populus x euramericana and of propolis by gas
chromatography-mass spectrometry. Proc R Scot Lond 1987;
B232: 249-72.
9 Zhen YS, Liu MS, Weber G. Effects of acivicin and dipyridamole
on hepatoma 3924A cells. Cancer Res 1983; 43: 1616-9.
10 Carmichael J, Degraff WG, Gazdar AF, Minna JD, Mitchell JB.
Evaluation of a tetrazolium-based semiautomated colorimetric
assay: assessment of chemosensitivity testing. Cancer Res
1987;47: 936-42.
11 Valduga G, Reddi E, Garbisa S, Jori G. Photosensitization of cells
with different metastatic potentials by liposome-delivered
Zn-(II)-pathalocyanine. Int J Cancer 1998; 75: 412-7.
12 Chiao C, Carothers AM, Grunbergers D, Solomon G, Preston GA,
Barrett JC. Apoptosis and altered redox state induced by caffeic
acid phenethyl ester (CAPE) in transformed rat fibroblast cells.
Cancer Res 1995; 55: 3576-83.
13 Hu ZB, Minden MD, McCulloch EA. Post-transcriptional
regulation of bcl-2 in acute myleoblastic leukemia: significance for
response to chemotherapy. Leukemia 1996; 10: 410-6.
14 Xu F, Zhen YS. (-)-Epigallocatechin-3-gallate enhances
anti-tumor effects of cytosine arabinoside on HL-60 cells. Acta
Pharmacol Sin 2003; 24: 163-8.
15 Kluck RM, McDougall CA, Harmon BV, Halliday JW. Calcium
chelators induce apoptosis: evidence that raised intracellular
ionized calcium is not essential for apoptosis. Biochim Biophys
Acta 1994; 1223: 247-54.
16 Tatton WG, Olanow CW. Apoptosis in neurodegenerative diseases:
the role of mitochondria. Biochim Biophys Acta 1999; 1410:
195-213.
17 Ichas F, Mazat JP. From calcium signaling to cell death: two
conformations for the mitochondrial permeability transition pore.
Switching from low to high conductance state. Biochim Biophys
Acta 1998; 1366: 33-50.
18 Ploemen JH, van Ommen B, de Haan A, Schefferlie JG, van
Bladeren PJ. In vitro and
in vivo reversible and irreversible
inhibition of rat glutathione-S-transferase isoenzymes by caffeic acid
and its 2-S-glutathionyl conjugate. Food Chem Toxicol 1993;
31: 475-82.
19 Shahrzad S, Bitsch I. Determination of some pharmacologically
active phenolic acids in juices by high-performance liquid
chromatography. J Chromatogr A 1996; 741: 223-31.
20 Slavin J, Jacobs D, Marquart L. Whole-grain consumption and
chronic disease: protective mechanisms. Nutr Cancer 1997; 27:
14-21.
21 Surh YJ. Transcription factors in the cellular signaling network
as prime targets of chemopreventive phytochemicals. Cancer
Treat Res 2004; 36: 275-86.
22 Liu J, Shen HM, Ong CN. Salvia
miltiorrhiza inhibits cell growth and induces apoptosis in human hepatoma
HepG2 cells. Cancer Lett 2000; 153: 85-93.
23 Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J,
et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from
mitochondria blocked. Science 1997; 275: 1129-32.
24 Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell
survival. Science 1998; 281: 1322-6.
25 Thornberry NA, Lazebnik Y. Caspase: enemies within. Science
1998; 281: 1312-6.
26 Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;
281: 1309-12.
27 Wallace DC. Mitochondrial disease in man and mouse. Science
1999; 283: 1482-8. |
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