J. Biomedical Science and Engineering, 2010, 3, 892-899
doi:10.4236/jbise.2010.39119 Published Online September 2010 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online September 2010 in SciRes. http://www.scirp.org/journal/jbise
Cytotoxic and inhibitory activity of ceramide on cancer cell
lines
Muthana Ibrahim Maleek
Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq.
Received 21 January 2010; revised 15 February 2010; accepted 15 February 2010.
ABSTRACT
The inhibitory activity of ceramide on cancer cells
was evaluated and its cytotoxicity on different cancer
cell lines was measured. Ceramide was separated fro-
m bovine brain and spinal cord, and extracted by or-
ganic solvents. The crude extract was purified by us-
ing silicic acid column. Detection and identification of
purified extract were carried out by using three ass-
ays: visualization, spectrophotometry and infrared.
Cytotoxic effect of different concentrations (7, 15, 30
and 60 μM) of ceramide on HEp-2, RD , AMGM5,
REFAM3 and AMN3 cancer cell lines was studied.
Results showed that ceramide at 30 μM imposed cy-
totoxic activity on all cancer cell lines especially on
AMGM5. Effect of ceramide at 30 μM on cell divi-
sion of human lymphocyte was also examined. Sig-
nificant reductions in mitotic and blast indices were
observed. In addition, No genotoxic effects or chrom-
osomal aberration were detected in lymphocyte chr-
omosomes when ceramide was tested in vitro.
Keywords: Ceramide; Cancer Cell Line; Cytotoxicity;
Genotoxicity; Chromosomal Aberration
1. INTRODUCTION
Cancer is a clonal disorder characterized by genetic in-
stability and shift in the control mechanism that govern
cell proliferation and differentiation [1,2]. It is a world-
wide health problem, with a geographical variation all
over the world [3].
Conventional cancer therapy is based on surgery, radi-
otherapy, chemotherapy or combinations of them. Gen-
erally, surgery and radiotherapy are preferred in local-
ized tumors and chemotherapy is used when cancer cells
are spread through the body [4]. The introduction of can-
cer chemotherapy in the 5th and 6th decades of the last
century has resulted in the development of curative the-
rapeutic interventions for patients with several types of
solid tumors and hematopoietic neoplasms. However, im-
portant obstacles were encountered in the use of chemo-
therapy that included toxicity to the normal tissues of the
body and the presence of mutations that confer resis-
tance to these chemotherapeutic agents [5]. Therefore,
cancer patients as well as many physicians began to re-
quest natural products for treatment due to their multiple
effects in treating the disease, relieving patient’s symp-
toms in addition to improved safety and lower cost [6].
Natural products have long been a fertile source of cure
for cancer, which is projected to become the major cause
of death in this century [7]. Therefore, a natural product,
ceramide, was tested for its inhibitory and cytotoxic ef-
fects on tumor cells.
The combination of sphingosine plus fatty acid is kno-
wn as ceramide (n-acylsphingosine), sphingosine or rela-
ted bases are important membrane components in animal
cells. They are present in large amounts especially in br-
ain and nerve tissue. Sphingolipids can be found in all
eukaryotic cells and the major phosphosphingolipid, sp-
hingomyelin, is very abundant in the outer leaflet of the
plasma membrane constituting of about 30% of all lipids
[8]. However, altogether over 300 sphingolipids with dis-
tinct head groups have been identified in eukaryotic cells.
In most mammalian cells, the structure is based on the
long chain of sphingosine base, which is subsequenttly
acylated to form ceramide (Figure 1) and more complex
sphingolipids are formed by the addition of polar head
groups to the C1 position of ceramide [9]. Sphingom-
yelins as phospholipids are formed when ceramide reacts
with phosphatidylcholine to form sphingomyelin plus
diacylglycerol. This occurs mainly in the Golgi appara-
tus and to a lesser extent in the plasma membrane. In
organelles involved in secretory and endocytic processes
sphingomyeline is restricted to the luminal aspect [10].
Ceramide was confirmed to function as a second me-
ssenger in several cellular processes, including apoptosis,
growth suppression, differentiation, transformation, proli-
feration, regulate cell-cell, cell-substrate interactions and

M. I. Maleek / J. Biomedical Science and Engineering 3 (2010) 892-899
893
Sphingosine
O +
║
CH3(CH2)14C---S----CoA Palmitoyl-CoA
↓
Ceramide (N-palmitoylsphingosine)
Figure 1. Formation of ceramide [10].
cell senescence [11]. In turn, aberrant metabolism of cer-
amide has been associated with inflammation, tumori ge-
nesis, diabetes, and neurodegenerative disorders [12,13].
Ceramide has emerged as a novel class of signaling mol-
ecules that also regulate phagocytosis [14]. The aim of
this study is to purify and characterize the ceramide that
was isolated from bovine brains and to study its ability
to inhibit tumor cells in vitro.
2. MATERIALS AND METHODS
2.1. Chemicals and Culture Media
Ciramide was separated and purified according to Dasgu-
pa et al. [15] and Dasgupta and Hogan, [16]. A modified
method of Bischel and Austin [17] was used to visualize
ceramide. The modification offered more sensitive and
stable color reaction in practice (down to 5-10 μg).
Silica gel G thin-layer chromatography and standard
solvent systems (chloroform: methanol: acetic acid; 95:
4.5:0.5) were employed to visualize ceramide. Chromat-
ograms were dried in a ventilated hood and sprayed with
the clorox reagent (5ml of clorox was added to 50 ml
benzene and then 5ml glacial acetic acid). The clorox re-
agent must be used immediately. After spraying, chro-
matograms were air dried in a hood to remove all unbou-
nd Cl2. In addition, paper chromatograms were also used
for ceramide visulization. They were rinsed twice with
tap water for 1-2 min duration, and allowed to air dry
until damp. They were then sprayed with benzidine re-
agent (0.5 g benzidine and 0.2 g of KI dissolved in 50 ml
of 50% ethanol) and then filtered. This solution was kept
out of direct light and used within 2h after preparation.
Alternatively, paper chromatograms may be dipped in the
benzidine reagent. Two more ways were also used for
visualization purposes; spectrophotometry assay and In-
frared assay, as described by Mc Murry [18].
Rosswell Park Memorial Institute (RPMI)-1640 med-
ium [19] was used as cell line growth medium. It was ki-
ndly provided by ICCMGR. All chemicals, solvents and
reagents were supplied by BDH (UK) unless otherwise
stated.
2.2. Cell Lines
1) Human larynx epidermoid carcinoma (HEp-2) cell
line was kindly provided by the Iraqi Center for Cancer
and Medical Genetics Research (ICCMGR). This human
cell line was originally obtained from a 57-year-old man
with a primary tumor of the larynx. Its biological and
chemical sensitivity has been very well documented
[20-23].
2) Rhabdomyosarcoma (RD) cell line was kindly pro-
vided by ICCMGR. This human cell line was derived
from a biopsy specimen obtained from a pelvic rhabdo-
myosarcoma of a 7-year-old
3) Ahmed-Majeed-glioblastoma-multiforme-2005 (AM-
GM5) cell line was kindly provided by Dr. A. Al-Sham-
ery from ICCMGR. This human cell line was obtained
from a human cerebral glioblastoma multiforme (GBM)
of a 72-year-old Iraqi male who underwent surgery for
intracranial tumor. Morphologic examination, immuno-
cytochemical staining, growth kinetics, and karyotypic
characteristics of this cell line were studied at ICCMGR
[A. Al-Shamery, personal communication]. The cultured
cells were spindle-like or polyhedral in shape. The po-
pulation doubling time was 28 hours. The chromosomal
number varied between 38 and 46, with mode chromo-
somal number of 42. In addition, chemotherapeutic drug
sensitivity was studied for AMGM5 cells at ICCMGR
and the cells appeared to be resistant to Cisplatin, Vin-
cristine and Etoposide.
4) Ahmed-Mohammed-Nahi-2003 (AMN3) cell line
was kindly provided by ICCMGR. This murine mamm-
ary adenocarcinoma cell line was derived from a sponta-
neous mammary adenocarcinoma of female BALB/c
mice [25].
5) Rat Embryo fibroblasts (REFAM3) cell line was
established and kindly provided by Dr. A. Al-Shamery
from ICCMGR. Cells of this normal murine cell line
were a mixture of fibroblastic and epithelial cells with
normal chromosomal picture [25].
Cell lines were maintained as described by Freshney
[19].
2.3. Assay of Ceramide Toxicity
Confluent monolayers were treated as in subculture .The
growth medium was decanted off and the cell sheet wash-
ed twice with PBS and trypsinized using trypsin-versene
(Gibco, Canada) [19]. When the cells are in exponential
growth, medium was removed and ceramide at varying
concentrations (7, 15, 30 and 60 μM) in serum free me-
dium (SFM) plus DMSO (1μl /ml) [26] were added to
the wells, five replicates were used for each concentra-
tion of ceramide. Twenty columns were used as control
(cells treated with SFM plus DMSO only). Plates were

M. I. Maleek / J. Biomedical Science and Engineering 3 (2010) 892-899
894
re-incubated at 37°C for 24 hrs and then medium was
replaced by 50μl of 0.01% crystal violet dye for 20 min-
utes, washed gently and allowed to dry [19]. Optical de-
nsity of each well was read by using a micro-ELISA rea-
der (Organon Teknika, Austria) at 492 nm transmitting
wavelength [19,27]. The percentage of inhibition was
calculated according to the following formula [28].
JBiSE

%
100 100
Inhibition
opticaldensity oftestwells
opticaldensity ofcontrolwells

 





that the extract was sphingolipid, as illustrated by Bisch-
el and Austin [17]. Since ceramide is one of the sphingo-
lipid classes [10] additional assays were carried out to
characterize it. A purified fraction which extracted by si-
licic acid was examined by spectrophotometer at a wide
ranges of wavelength (between 200-1100 nm). One peak
at wavelength 326nm with absorbance 1.481 was noticed
(Figure 2). Examination of the purified compound by
infrared instrument showed many peaks for many infra-
red absorption frequencies (Figure 3) and each value of
absorption was referred to a functional group of test co-
mpound as follow: [748.34 (O-disubstituted), 964.34
(RCH = CH2), 1126.35(-C—C-), 1280.65 (-O-C-ether),
1380.94-1458.8(CH2), 1650(C = C), 1728.10 (C = O),
2854.45(C-H strong), 2923.88(C-H alkane), 2970-
3140(N-H) and 3363.62 (-O-H alcohol)]. When these
functional groups were compared with the chemical co-
mponents of ceramide, the results showed that the extra-
cted compound can be identified as ceramide according
to Mc Murry [18].
3. RESULTS AND DISCUSSION
Ceramide was separated from bovine brain and spinal
cord. The detection and identification of purified extract
were examined by using different assays. Using the ben-
zidine spray visualization assay ceramide was detected
by the virtue of the reaction of their secondary amide
group belongs to the test compound with CL-substituted.
After that the result of combination with benzidine spray
was observed, blue product was formed, which means
100.0
(
NM/Div.
)
NM
1100.0
326.0 1.481 722.0 -0.000
λ ABS λ ABS
- - PEAK- - - -QUALITY- -
*** PEAK-PICK ***
200.0
1.07 0/02 .00 1100.0 NM 0.001 A
+0.00 A
0.500
(A/Div.)
+0.25
A
Figure 2. Spectrogram of extracted ceramide.
100
95
90
85
80
75
%T
3000 1000 4000 2000
1/cm
3933.2
6
2854.4
5
1728.1
0
1458.08
1380.9
4
1260.65
5
1126.3
5
2923.8
8
964.3
4
748.3
3
Comments: No. of Scans; 10, Resolution; 16/[1/cm], Apodization; Happ-Genzel
Figure 3. Infrared absorption frequencies assay for ceramide extract.

M. I. Maleek / J. Biomedical Science and Engineering 3 (2010) 892-899
895
Effect of different concentrations of purified ceramide
on HEp-2 cells was studied (Figure 4). There was a re-
duction in cell viability in almost all tested concentra-
tions of ceramide. The percentage of inhibition was
13.2% at 15 µM of ceramide. Similarly a percentage of
inhibition of 13.1% was obtained when cells treated with
30 µM of ceramide. But when the highest ceramide (60
µM) a percentage of inhibition of 12.5% was obtained.
However, 7 µM of ceramide imposed the lowest cyto-
toxic efficacy on HEp-2 cells, where the percentage of
inhibition was 1%.
Different concentrations of ceramide influenced the
viability of RD cells as shown in Figure 5. Low concen-
trations of ceramide (7 and 15 µM) showed low toxic eff-
ect on the viability of RD cells, giving percentage of inh-
ibition of 6.7% and 7.5%, respectively. However, 30 µM
of ceramide produced higher toxic effect of 26.3%, fol-
lowed by a reduction to 18.8% at 60 µM.
Figure 4. Effect of different concentration of ceramide on viability of
HEp-2 cell line ( P ≤ 0.05).
Figure 5. Effect of different concentration of ceramide on viability of
RD cell line ( P ≤ 0.05).

M. I. Maleek / J. Biomedical Science and Engineering 3 (2010) 892-899
896
AMGM5 cells underwent significant toxic changes
towards different concentrations of ceramide (Figure 6).
The percentage of AMGM5 cells inhibition was gradu-
ally increased (21, 47 and 79%) when treated with 7, 15
and 30µM of ceramide, respectively. At 60 µM of cera-
mide the percentage of inhibition decreased to (50%). It
is apparent that AMGM5 cells treated with 30 µM of
ceramide showed suffered a significant reduction in cells
viability.
The cytotoxic effect of different concentration of cera-
mide on viability of AMN3 cells were illustrated in Fi-
gure 6. The percentage of inhibition was gradually in-
creased (3.06, 10.7, 13.26 and 19.13%) at concentrations
of the ceramide 7, 15, 30 and 60 µM, respectively.
The cytotoxicity effects of different concentrations of
ceramide on normal cells line REFAM3 were illustrated
in Figure 7. Both concentrations of ceramide 30 and 60
µM achieved high percentage of inhibition of 16.7 and
15.5%, respectively. While cells treated with low con-
centrations of ceramide (7 and 15 µM) showed low level
of toxicity giving percentage of inhibition of 11.3 and
3.3%, respectively.
According to the above findings, it can be concluded
that ceramide has inhibitory activity on almost all cell
lines used in this study. As shown in Figure 8 that cera-
mide at 30 µM has a clear cytotoxic activity on all cell
lines used, especially on AMGM5 cells; the mean of
growth inhibition was 49.4% (Figure 9). This may be
0
10
20
30
40
50
60
70
80
90
7153060
% Inhibition
Con centratio n of cer am id e (uM)
Figure 6. Effect of different concentration of ceramide on viability of AMGM5 cells (P ≤ 0.05).
0
5
10
15
20
25
7 153060
% Inhibition
Concentration of ceramide (uM)
Figure 7. Effect of ceramide on viability of AMN3 cells ( P ≤0.05).

M. I. Maleek / J. Biomedical Science and Engineering 3 (2010) 892-899
897
Figure 8. Effect of different concentration of ceramide on viability of REFAM3 cells ( P ≤ 0.05).
Figure 9. Relationship between growth inhibition of cell lines at different concentrations of ce-
ramide ( P ≤ 0.05).
due to the cellular nature of AMGM5 cell line. As men-
tioned earlier that AMGM5 is a human cell line origi-
nated from a human cerebral glastoblastoma multiforme
(GBM). In addition, ceramide was proved to bind vig-
orously to CD95 receptors on cerebral cells, thus this
specificity of engagement may initiate multiple signaling
pathways that lead to activate caspases which is respon-
sible for apoptosis [29-31]. This conclusion agreed with
that of Seumois et al. [32] who recently demonstrated on
the de novo generation that ceramide contributes to spo-
ntaneous neutrophil apoptosis via caspase activation. On
the other hand, the addition of ceramide to REFAM3, a
normal cell line which is a mixture of fibroblastic and
epithelial cells with normal chromosomal picture,
showed low cytotoxic effect of ceramide (Figure 9 and
10). This may be attributed to the ability of these cells to
generate ceramide-1-phosphate (cer-1-p) by the action of
ceramide kinase [33]. Subsequently, cer-1-p in plasma
membrane can be hydrolyzed by phosphatases [34]. Re-
cently, Rile et al. [35] reported that cer-1-p can also be

M. I. Maleek / J. Biomedical Science and Engineering 3 (2010) 892-899
898
Figure 10. Mean of growth inhibition of five cell lines after treated with ceramide (P ≤ 0.01).
formed intracellularly.
It can be concluded that ceramide has cytotoxic prop-
erties on cancer cell while it is much less toxic to normal
cells. Generally, 30 µM of this compound gave the hig-
hest affect in almost all cancer cell lines tested.
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