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Antiproliferative activity of eugenol and curcumin related
biphenyls on malignant melanoma cell lines
Marina Pisano, Monica Loi, Maria Giovanna Tilocca, Giuseppe Palmieri, Gabriella Pagnan§, Davide Fabbri,
Maria Antonietta Dettori, Giovanna Delogu, Mirco Ponzoni§ and Carla Rozzo.
Istituto di Chimica Biomolecolare-CNR, Li Punti-Sassari, Italy; §Laboratorio di Oncologia, Ospedale “G.Gaslini”,Genova, Italy.
Abstract
Background: Malignant Melanoma (MM) is one of the most aggressive cancer and its
incidence and mortality rates are highly increased during the last decades in fair skin
populations. Chemotherapeutics currently in use are still unsatisfactory therefore the
search for novel therapies is warranted. Eugenol and curcumin, main components of
several spices, have both been described as potential anticancer agents. We tested
several eugenol and curcumin-related compounds for their capability to inhibit cell
growth on primary MM cell lines. Material and methods: Viability and cytotoxicity
assays together with dose and time-response assays have been carried out on MM cell
lines to compare the antitumour activity of both eugenol and curcumin to that of 11
related compounds (monomeric or biphenyl structures). Cultured fibroblasts from
healthy donors have been used as controls. TUNEL assays have been performed to
assess apoptosis triggered by some of the treatments. Results: Among the eugenol-
related biphenyls, dibromo-dehydrodieugenol (S7) showed good antiproliferative
activity, specific on MM cells, being its enantiomeric form (S7-S) the most active with
IC50 ranging around 20-30M, and triggering apoptosis (M.Pisano et al, Mol Cancer 6:8,
2007). We also tested five curcumin-related hydroxylated biphenyls (D2-D6) on MM
cells to assess their antiproliferative activity in comparison with that of curcumin, which
itself strongly inhibited MM cells growth, and that of the S7-S biphenyl. These
preliminary experiments suggest that curcumin-related biphenyls are the most active in
inhibiting MM cells growth. In particular the ,-unsaturated keton (D6) shows
antiproliferative activity at concentrations lower than curcumin (IC50 around 1-2M) on
MM cells, not affecting normal fibroblasts proliferation rate. Work is on running to define
doses and time of D6 action on MM cells and to investigate on its pro-apoptotic activity.
Conclusions: Our results indicate eugenol and curcumin related biphenyls as good
leads to develop new therapeutic agents against MM. Their activity should be
investigated on in vivo melanoma models to assess the real anticancer effectiveness on
such tumour.
Figure 1. Effect of eugenol (S1)
and eugenol-derivatives (S2-S7)
on MM cell lines proliferation.
Three MM cell lines were grown
up to 6 days in presence of 100M
of each of the 7 compounds S1-
S7. Even if the sensibility of each
cell line is different the
antiproliferative activity increases
from the monomeric forms (S1–
S4) to the biphenyl forms (S5 and
S7) being the S7 treatment the
most effective (~100% of cell
growth inhibition).
IC50 M (96hrs)
Cell lines
S7 raceme
S7-R
S7-S
PNP 46,0 41 29
WM
38,5
36
27
GR 30,0 31,5 23
Table 1
Table 1. Enantiopure form S7-S
is more active than both S7-R
and their racemic mixture. More
dose-dependence assays were
carried out focusing on the
antiproliferative action of S7 and
its enantiopure forms (S7-S and
S7-R) showing that the S7-S
enantiomeric form was the most
active on inhibiting melanoma cell
proliferation. Table 1 shows IC50
values for the three S7 forms.
Fig. 2 Wash-out S7-S 50M
0
20
40
60
80
100
120
6 12 18 24 48
cell growth (%)
WM
PNP
FibroNA
Figure 2. Anticancer specificity of S7-S.
To simulate the S7-S activity in vivo and better define the action
time window of such compound MM cells were grown up to 48hrs
by treating them with 50M S7-S and washing it out after short
exposures (from 6 to 48hrs). The figure shows that a mono-dose of
S7-S for 48hrs is sufficient to inhibit MM cell growth up to 80% with
no significant effect on normal fibroblast cells growth.
Figure 3. Effects of S7-S on triggering MM cells apoptosis.
To determine whether the S7-S antiproliferative action on MM cells
occurred via induction of apoptosis, we performed TUNEL assays to
measure DNA fragmentation. A small, but consistent proportion of
TUNEL-positive melanoma cells (15-20% apoptotic fraction) was
detected after 24hrs of treatment with 50µM S7-S while no sign of
apoptosis features were observed in human fibroblasts.
Figure 4. Effect of curcumin (D1) and curcumin-related
biphenyls (D2-D6) on MM cell proliferation.
Two MM cell lines were grown up to 6 days in presence of 100M
curcumin or one of the following biphenyls derivatives. The -keto
acid O-Me-protected (D2) and the -ketoacid O-Bn-protected (D4)
did not show high proliferation inhibition, while the ,-unsaturated
ester (D3) showed a 40-50% growth inhibition, but the -keto ester
(D5) and the ,-unsaturated keton (D6) were the most active with a
cell growth inhibition comparable to curcumin (~100%).
Table 2. The ,-unsaturated keton D6 is the most active biphenyl curcumin-related.
Dose-dependent experiments were carried out using five different MM cell lines in order to evaluate the real
antiproliferative effect for each of the curcumin-related structures (D2-D6). MM cells were grown up to five days with
variable concentrations of each curcumin derivative. Table 2 shows a summary of the results revealing for the ,-
unsaturated keton D6 IC50 values around 1M.
IC50 D1-D6 24 hrs
0
10
20
30
40
50
60
70
80
90
100
110
120
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80
concentrations (M)
cell growth (%)
D1 LB
D1 LCP
D6 LB
D6 LCP
Fig.5
Figure 5. Comparison of curcumin (D1)
and the -unsaturated keton (D6)
antiproliferative activity: IC50.
MM cells were grown up to 24hrs in the
presence of D1 or D6 at scalar
concentrations from 1M up to 80M. The
figure shows the higher antiproliferative
effect of D6 on two MM cell lines, with IC50
values as low as 2M (LB) and 4M (LCP)
while D1 shows IC50 of 14M and 27M
respectively.
Figure 6. Comparison of D1 and D6 antiproliferative activity: Wash-out.
Wash-out experiments were carried out with a 5M single dose of D1 or D6 on two MM
cell lines (LB and PNP) and a normal fibroblast cell line (BJ) for 6, 12 and 24hrs. Growth
rate was measured after 24hrs (A) or 72hrs (B). One 6hrs treatment was sufficient for D6
(but not for D1) to inhibit MM cells proliferation up to 90% after 24 hrs, having a small
effect (10-20%) on BJ normal fibroblasts. The early effect of D6 was even more evident
when cells were let grown up to 72 hrs showing the irreversibility of D6 action.
H
3
CO
HO
H3CO
H3CO
H
3
CO
HO
H3CO
H3CO
H3CO
HO
H3CO
HO
H3CO
H3CO
H3CO
H3CO
H3CO
HO
H3CO
HO Br
Br
Fig.1 Cytotoxicity S1-S7
0
20
40
60
80
100
120
S1 S2 S3 S4 S5 S6 S7
treatments (100 M)
cell growth (%)
PNP
WM 266
GR
Work in progress: manuscript concerning synthesis and characterization of the curcumin-related compounds is under preparation. Preliminary results suggest that D6
antitumoral activity may be related to apoptosis induction. Efforts should be spent to define in deep the molecular mechanism of this compound. Further investigation of D6 in
mouse cancer models will contribute to additional understanding its in vivo activity toward malignant cells and its effects on normal tissues.
24 hrs
72 hrs
PNP
LB
BJ
D6 5M
D1 5M
Fig.6
0
10
20
30
40
50
60
70
80
90
100
6 h 12 h 24 h
treatment (hrs)
cell growth (%)
0
10
20
30
40
50
60
70
80
90
100
6 h 12 h 24 h
treatment (hrs)
cell growth (%)
0
10
20
30
40
50
60
70
80
90
100
6 h 12 h 24 h 72 h
treatment (hrs)
cell growth (%)
0
10
20
30
40
50
60
70
80
90
100
6 h 12 h 24 h 72 h
treatment (hrs)
cell growth (%)
A
B
1,2nd> 10080> 100PNP
1,2513ndndndLCP
111,2> 100> 100> 100LB
1,88,2> 1005851WM
1,613> 1003847CN
D6D5D4D3D2Cell lines
1,2nd> 10080> 100PNP
13ndndndLCP
111,2> 100> 100
>
100
LB
8,2> 1005851WM
13> 1003847CN
D6D5D4D3D2
IC50 M (96hrs)
Table 2
100m
Control
50M S7-S
GR Sbcl2 Fibroblasts
Fig. 3
O
O
CH3O OH
H3CO OH
treatment (hrs)
Cytotoxicity D1-D6
0
20
40
60
80
100
120
D1 D2 D3 D4 D5 D6
treatments (100 uM)
cell growth (%)
PNP
GR
Fig. 4
D1
