Enhanced chemopreventive activity of hydroxytyrosol on HL60 and HL60R cells by chemical conversion into thio derivatives

Article abstract of DOI:10.1016/j.ejps.2012.12.028

Thio derivatives of hydroxytyrosol containing thiol, thioacetate and disulfide functionalities were synthesized from natural hydroxytyrosol (3,4-DHPEA) via 3,4-dihydroxyphenethyl halides. These compounds, containing the combination of catechol moiety and

Full text of DOI:10.1016/j.ejps.2012.12.028

European Journal of Pharmaceutical Sciences 48 (2013) 790–798
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Enhanced chemopreventive activity of hydroxytyrosol on HL60 and HL60R cells
by chemical conversion into thio derivatives
b
Maria Vittoria Sepporta a,1, M. Ángeles López-García b,1, Roberto Fabiani a, , Inés Maya ,
⇑
José G. Fernández-Bolaños b,
⇑
a Dipartimento di Specialità Medico Chirurgiche e Sanità Pubblica, Sezione di Epidemiologia Molecolare e Igiene Ambientale, Università di Perugia, Via del Giochetto, 06126
Perugia, Italy
b Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, c/Profesor García González 1, 41012 Sevilla, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Thio derivatives of hydroxytyrosol containing thiol, thioacetate and disulfide functionalities were synthe-
sized from natural hydroxytyrosol (3,4-DHPEA) via 3,4-dihydroxyphenethyl halides. These compounds,
containing the combination of catechol moiety and divalent sulfur functions, were tested for the pro-
apoptotic and anti-proliferative activities on both parental HL60 and multi-drug resistant HL60R cells.
It was found that all synthesized compounds were more effective than 3,4-DHPEA in inducing apoptosis
on HL60R cells, and that the hydroxytyrosol disulfide was the most active pro-apoptotic and anti-prolif-
erative compound on both HL60 and HL60R cells. Different from 3,4-DHPEA, all thio derivatives of
hydroxytyrosol induced apoptosis by a mechanism not involving the release of H2O2 in the culture med-
ium. The data on HL60R cells suggest that these compounds could be able to reverse the resistance
toward the most common drugs in cancer therapy.
Received 1 November 2012
Received in revised form 18 December 2012
Accepted 20 December 2012
Available online 9 January 2013
Keywords:
Apoptosis
Proliferation
Sulfur compounds
Hydroxytyrosol
HL60 cells
Ó 2013 Elsevier B.V. All rights reserved.
Multi drug resistance (MDR)
1. Introduction
in a high concentration range (100–800 lM) and were mostly
dependent upon the ability of 3,4-DHPEA to induce the accumula-
tion of hydrogen peroxide (H2O2) in the cell culture medium (Fabi-
ani et al., 2009).
Hydroxytyrosol 1 (3,4-dihydroxyphenylethanol: 3,4-DHPEA) is
the main ortho-diphenol present exclusively and abundantly in ol-
ive oil both as a free compound and linked to the dialdehydic form
of decarboxymethyl elenolic acid (3,4-DHPEA-EDA), and also as an
isomer of oleuropein aglycon (3,4-DHPEA-EA) (Servili and Monted-
oro, 2002). In the past few years several biological properties of
3,4-DHPEA have been deeply investigated (Fernández-Bolaños
et al., 2008, 2012) to explain the epidemiological data indicating
an inverse correlation between olive oil consumption and the risk
of some degenerative diseases, in particular coronary heart dis-
eases and cancer (Covas, 2008; Pelucchi et al., 2011). Regarding
the cancer chemopreventive activities, it has been shown that
3,4-DHPEA is able to inhibit both initiation and promotion/pro-
gression phases of carcinogenesis by preventing the DNA damage
induced by different genotoxic molecules (Fabiani et al., 2008)
and by inhibiting the proliferation and inducing the apoptosis in
different tumors cell lines, respectively (Fabiani et al., 2002). It
should be noted, however, that these last effects were evident only
Vegetables of genus Allium and Cruciferous such as garlic,
onions, scallions, chives, leeks and broccoli have shown potential
chemopreventive properties, which have been attributed to the
presence of high levels of organosulfur compounds (Battin and
Brumaghim, 2009; Nagini, 2008). Such compounds have shown a
role in the prevention of cancer, cardiovascular, neurodegenera-
tive, and inflammatory diseases (Tapiero et al., 2004) due to their
antioxidant and antiatherosclerotic properties (Vazquez-Prieto
and Miatello, 2010). Recently, it has been reported that diallyl
disulfide exerts an antiproliferative activity in cancer cells (Wong
et al., 2010; Kim et al., 2010). Thio derivatives of hydroxytyrosol
such as 3,4-dihydroxyphenethyl methyl sulfide, and their sulfox-
ide, sulfone and methylsulfonium derivatives, have been described
as dopamine agonists (Wallace et al., 1989). Bis(3,4-hydroxyphen-
ethyl) sulfide has shown to be a therapeutic agent useful in the
nephritis treatment (Hosegawa et al., 1998). Allyl 3,4-hydroxy-
phenethyl disulfide has been used in the preparation of a dopa-
mine synthetic receptor (Takeuchi et al., 2006).
⇑
Corresponding authors. Tel.: +39 075 5857336; fax: +39 075 5857328
In this paper, we describe the synthesis of hydroxytyrosol deriv-
atives containing thiol, thioacetate and disulfide functionalities in
order to test, for the first time, if the combination of the catechol
(R. Fabiani), tel.: +34 954550996; fax: +34 954624960 (J.G. Fernández-Bolaños).
E-mail addresses: fabirob@unipg.it (R. Fabiani), bolanos@us.es (J.G. Fernández-
Bolaños).
1
These authors contributed equally to this work.
0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejps.2012.12.028

M.V. Sepporta et al. / European Journal of Pharmaceutical Sciences 48 (2013) 790–798
791
moiety and sulfur containing functions results in an improvement
of the pro-apoptotic and anti-proliferative activities shown by 3,4-
DHPEA. We have determined the ability of 3,4-DHPEA and its thio
derivatives to induce cell death, to inhibit proliferation and to
cause H2O2 accumulation in the culture medium on both, acute
myeloid leukemia HL60 cell line and its multidrug resistant
(MDR), P-glycoprotein overexpressing, variant HL60R. As it is
known that the HL60R cells have a reduced sensitivity to the cell
death induction by treatment with the most common chemother-
apeutic drugs, such as daunorubicin and cytosine arabinoside, it is
of special importance to discover new compounds active on these
cell lines (Notarbartolo et al., 2002).
concentrated. The residue was purified by column chromatography
(EtOAc–hexane 1:5 ? 1:3) to give 3 as a colourless solid. Yield
356 mg, 69%; RF 0.42 (EtOAc–hexane 1:2); m.p.: 110–112 °C; 1H-
RMN (300 MHz, CD3OD): d (ppm) 6.69 (d, 1H, J5,6 = 8.0 Hz, H-6),
6.64 (d, 1H, J3,5 = 2.0 Hz, H-3), 6.52 (dd, 1H, H-5), 3.30 (t, 2H,
J1,2 = 7.7 Hz, CH2I), 2.97 (t, 2H, CH2Ar); 13C-RMN (75.5 MHz, CD3-
OD): d (ppm) 146.2, 145.0 (C-1, C-2), 133.9 (C-4), 120.7 (C-5),
116.4 (C-3, C-6), 41.0 (CH2Ar), 6.7 (CH2I); IR mmax 3486, 3332,
3033, 2949, 1732, 1601, 1515, 1454, 1346, 1275 cmÀ1; EIMS m/z
264 ([M]+, 32%); HREIMS m/z calcd for [M]+ C8H9IO2: 263.9647,
found: 263.9648.
2.4. 1,2-Diacetoxy-4-(2-chloroethyl)benzene (4)
2. Materials and methods
A solution of 2 (55 mg, 0.32 mmol) in a mixture of Ac2O–Py 1:1
(2.0 mL) was kept at 4 °C for 24 h. Then, the solvent was removed
under vacuum, co-evaporating with toluene and ethanol, and the
residue purified by column chromatography (EtOAc–hexane 1:5),
which gave 4 as a colourless oil. Yield 65 mg, 79%; RF 0.46
(EtOAc–hexane 1:2); 1H-RMN (300 MHz, CDCl3): d (ppm) 7.13 (d,
1H, J5,6 = 8.2 Hz, H-6), 7.09 (dd, 1H, J3,5 = 2.1 Hz, H-5), 7.06 (d, 1H,
H-3), 3.69 (t, 2H, J1,2 = 7.4 Hz, CH2Cl), 3.05 (t, 2H, CH2Ar), 2.28,
2.27 (2s, 6H, 2CH3CO); 13C-RMN (75.5 MHz, CDCl3): d (ppm)
168.4, 168.3 (2CH3CO), 142.1, 141.1 (C-1, C-2), 137.0 (C-4), 127.1
(C-5), 123.9 (C-3), 123.5 (C-6), 44.5 (CH2Cl), 38.5 (CH2Ar), 20.7
(2CH3CO); IR mmax 3369, 3220, 2962, 2919, 2380, 2346, 1764,
1502, 1428, 1371, 1259, 1201, 1177, 1104, 1007 cmÀ1; EIMS m/z
256 ([M]+, 5%); HREIMS m/z calcd for [M]+ C12H1335ClO4:
256.0502, found: 256.0504.
2.1. General chemical procedures
1H (300 and 500 MHz) and 13C (75.5 and 125.8 MHz) NMR spec-
tra were recorded on Bruker Avance-300 and Avance-500 spec-
trometers. The assignments of 1H and 13C signals were confirmed
by homonuclear COSY and heteronuclear 2D correlated spectra,
respectively. Mass spectra (EI) were recorded on a Micromass
AutoSpec-Q mass spectrometer. IR spectra were recorded on Jasco
FT/IR-4100 spectrometer. TLC was performed on aluminium pre-
coated sheets (E. Merck Silica Gel 60 F254), spots were visualized
by UV light and by charring with 10% H2SO4 in EtOH. Column
chromatography was performed using E. Merck Silica Gel 60
(40–63 lm). All reactions were carried out in dry solvents, under
argon and in darkness. 3,4-DHPEA used as starting material was
provided as an ethanol/water 1:4 solution at 6.3% (purity degree
97%) obtained from olive mill wastewaters (Fernández-Bolaños
et al., 2002).
2.5. 1,2-Diacetoxy-4-(2-iodoethyl)benzene (5)
A solution of 3 (300 mg, 1.14 mmol) in a mixture of Ac2O–Py 1:1
(5.0 mL) was kept at 4 °C for 24 h. Then, the solvent was removed
under vacuum, co-evaporating with toluene and ethanol, and the
residue purified by column chromatography (EtOAc–hexane 1:4),
which gave 5 as a yellowish oil. Yield 267 mg, 75%; RF 0.51
(EtOAc–hexane 1:2); 1H-RMN (300 MHz, CDCl3): d (ppm) 7.13 (d,
1H, J5,6 = 8.2 Hz, H-6), 7.07 (dd, 1H, J3,5 = 2.0 Hz, H-5), 7.03 (d, 1H,
H-3), 3.34 (m, 2H, J1,2 = 7.7 Hz, CH2I), 3.17 (m, 2H, CH2Ar), 2.28,
2.28 (2s, 6H, 2CH3CO); 13C-RMN (75.5 MHz, CDCl3): d (ppm)
168.3, 168.3 (2CH3CO), 142.1, 141.0 (C-1, C-2), 139.4 (C-4), 126.5
(C-5), 123.6 (C-6), 123.4 (C-3), 39.8 (CH2Ar), 20.7 (2CH3CO), 4.3
(CH2I); IR mmax 3369, 3229, 2962, 2938, 2350, 1759, 1589, 1502,
1429, 1366, 1255, 1192, 1113, 1012 cmÀ1; EIMS m/z 348 ([M]+,
9%); HREIMS m/z calcd for [M]+ C12H13IO4: 347.9859, found:
347.9865.
2.2. 4-(2-Chloroethyl)benzene-1,2-diol (2)
To a solution of hydroxytyrosol (103 mg, 0.67 mmol) in dry CH3-
CN (5 mL), PPh3 (238.3 mg, 0.91 mmol) and CCl4 (171 lL,
1.77 mmol) were added. The reaction mixture was stirred at r.t.
for 10 h. Then, the solvent was removed under vacuum and the res-
idue was suspended in CH2Cl2 and washed with water. The
collected organic layer was dried (MgSO4) and concentrated. The
residue was purified by column chromatography (EtOAc–hexane
1:5) to give 2 as a colourless solid. Yield 67 mg, 60%; RF 0.39
(EtOAc–hexane 1:2); m.p.: 102–104 °C; 1H-RMN (300 MHz, CD3-
OD):
d (ppm) 6.69 (d, 1H, J5,6 = 8.0 Hz, H-6), 6.66 (d, 1H,
J3,5 = 2.0 Hz, H-3), 6.54 (dd, 1H, H-5), 3.64 (t, 2H, J1,2 = 7.5 Hz, CH2-
Cl), 2.87 (t, 2H, CH2Ar); 13C-RMN (75.5 MHz, CD3OD): d (ppm)
146.2, 145.1 (C-1, C-2), 131.3 (C-4), 121.2 (C-5), 116.9 (C-3),
116.4 (C-6), 46.1 (CH2Cl), 39.8 (CH2Ar); IR mmax 3448, 3321, 2953,
2924, 2865, 1617, 1531, 1451, 1378, 1280, 1273 cmÀ1; EIMS m/z
172 [M]+, 54%); HREIMS m/z calcd for [M]+ C8H935ClO2: 172.0291,
found: 172.0296.
2.6. S-2-(3,4-Diacetoxyphenyl)ethyl ethanethioate (6)
To a solution of 4 or 5 (1 mmol) in butanone (5 mL), potassium
thioacetate (1.4 mmol) was added. The reaction mixture was stir-
red at reflux for 2–6 h. Then, the solvent was removed under vac-
uum and the residue was suspended in EtOAc and washed with
water. The water layer was extracted with EtOAc and the collected
organic layers were then dried (MgSO4) and concentrated. The res-
idue was purified by column chromatography (EtOAc–hexane 1:6)
to give 6 as a reddish solid. Yield 142 mg, 48% from 4; 225 mg, 76%
from 5; RF 0.36 (EtOAc–hexane 1:2), m.p.: 39–41 °C; 1H-RMN
(300 MHz, CDCl3): d (ppm) 7.11–7.05 (m, 3H, H-3, H-5, H-6), 3.09
(m, 2H, CH2S), 2.85 (m, 2H, CH2Ar), 2.33 (s, 3H, CH3COS), 2.28,
2.27 (2s, 6H, 2CH3CO); 13C-RMN(75.5 MHz, CDCl3): d (ppm) 195.6
(SCOCH3), 168.4, 168.3 (2CH3CO), 142.0, 140.7 (C-1, C-2), 138.9
(C-4), 126.8 (C-5), 123.6 (C-6), 123.4 (C-3), 35.4 (CH2Ar), 30.7 (CH3-
COS), 30.1 (CH2S), 20.7 (2CH3CO); IR mmax 3374, 3316, 3239, 2977,
2919, 1774, 1682, 1502, 1424, 1366, 1264, 1192, 1133, 1104, 1012,
2.3. 4-(2-Iodoethyl)benzene-1,2-diol (3)
To a solution of hydroxytyrosol (310 mg, 2.01 mmol) in dry THF
(5 mL), PPh3 (510 mg, 1.95 mmol), imidazole (265 mg, 3.89 mmol),
I2 (741 mg, 2.92 mmol) and 4 Å molecular sieve were added. The
reaction mixture was stirred under argon atmosphere at reflux
for 3 h. Then, the reaction mixture was filtered and the solvent
was removed under vacuum. The residue was suspended in EtOAc,
saturated aqueous solutions of NaHCO3 (5 mL) and of Na2S2O3
(5 mL) were added and the mixture was stirred for 5 min. The or-
ganic layer was separated and the aqueous layer was extracted
with EtOAc. The collected organic layers were dried (MgSO4) and

792
M.V. Sepporta et al. / European Journal of Pharmaceutical Sciences 48 (2013) 790–798
896, 623 cmÀ1; EIMS m/z 296 ([M]+, 1%); HREIMS m/z calcd for [M]+
C14H16O5S: 296.0718, found: 296.0714.
Scotland). 3,4-DHPEA was obtained from Cayman Chemicals Ltd.
(United States) as ethanol solution at a concentration of 320 mM.
The synthesized compounds, hydroxytyrosol derivatives, were dis-
solved in DMSO at a concentration of 10 mM. The compounds were
stored at À20 °C in the dark and diluted in RPMI 1640 medium to
the desired concentration just before use. All the solutions were
2.7. S-2-(3,4-Dihydroxyphenyl)ethyl ethanethioate (7)
To a solution of 6 (100 mg, 0.34 mmol) in MeOH (2 mL), aque-
ous 2 N HCl (1 mL) was added and the reaction mixture was stirred
under argon atmosphere and in the darkness at r.t. for 12 h. Then,
the reaction mixture was neutralized to pH 5 with a saturated
aqueous solution of NaHCO3 and the solvent was removed under
vacuum. The residue was purified by column chromatography
(EtOAc–hexane 1:5 ? 1:3) to give 7 as a reddish oil. Yield 58 mg,
86%; RF 0.35 (EtOAc–hexane 1:2); 1H-RMN (300 MHz, CDCl3): d
(ppm) 6.79 (d, 1H, J5,6 = 8.1 Hz, H-5), 6.74 (d, 1H, J2,6 = 1.9 Hz, H-
2), 6.62 (dd, 1H, H-6), 5.70 (br. s, 2H, 2OH), 3.06 (m, 2H, CH2S),
2.71 (m, 2H, CH2Ar), 2.34 (s, 3H, CH3COS); 13C-RMN (75.5 MHz,
CDCl3): d (ppm) 197.3 (SCOCH3), 143.7, 142.4 (C-3, C-4), 133.0
(C-1), 121.1 (C-6), 123.6 (C-2), 123.4 (C-5), 35.0 (CH2Ar), 30.8
(CH2S), 30.8 (CH3CO); IR mmax 3335, 2924, 2850, 2375, 2346,
1715, 1652, 1604, 1517, 1444, 1366, 1279, 1250, 1192, 1114,
1051 cmÀ1; EIMS m/z 212 ([M]+, 6%); HREIMS m/z calcd for [M]+
C10H12O3S: 212.0507, found: 212.0506.
sterilized by filtration on 0.22 lm filters (Celbio S.r.l., Milan, Italy).
All other reagents were purchased from Sigma unless differently
specified (Sigma–Aldrich Co., Ltd., Irvine, UK). Human promyelo-
cytic leukemia cells (HL60) were obtained from the American Type
Culture Collection (Rockville, MD, USA) while its multidrug resis-
tant (MDR) variant (HL60R) were provided by Dr. Marilena Cre-
scimanno (University of Palermo). The properties of the HL60R
cells were described previously (Grimaudo et al., 1998). The cells
were cultured in complete medium (RPMI 1640 supplemented
with 10% FCS, 2.0 mM
L-glutamine, 100 U/ml penicillin and
100 U/ml streptomycin) at 37 °C and 5% CO2 in a humidified atmo-
sphere and seeded every 4–5 days at a proper density as suggested
by the supplier.
2.11. Cell treatments and proliferation assay
HL60 and HL60R cells were seeded at a density of 0.2 Â 106/mL
in the complete medium on 24-well plates (Celbio S.r.l., Milan,
Italy) and treated with the different compounds at 37 °C and 5%
2.8. 4-(2-Sulfanylethyl)benzene-1,2-diol (8)
To a solution of 6 (100 mg, 0.34 mmol) in MeOH (2 mL), aque-
ous 2 N HCl (1 mL) was added and the reaction mixture was stirred
under argon atmosphere and in the darkness at 40 °C for 34 h.
Then, the reaction mixture was neutralized to pH 5 with a satu-
rated aqueous solution of NaHCO3 and the solvent was removed
under vacuum. The residue was purified by column chromatogra-
phy (EtOAc–hexane 1:5) to give 8 as a yellowish solid. Yield
57 mg, 98%; RF 0.30 (EtOAc–hexane 1:2), m.p.: 77–78 °C; 1H-RMN
(300 MHz, (CD3)2CO): d (ppm) 6.74 (d, 1H, J5,6 = 8.0 Hz, H-6), 6.71
(d, 1H, J3,5 = 2.0 Hz, H-3), 6.55 (dd, 1H, H-5), 3.06 (2br. s, 2H,
2OH), 2.72 (m, 4H, CH2S, CH2Ar), 1.64 (t, 1H, J2,SH = 7.2 Hz, SH);
13C-RMN (75.5 MHz, (CD3)2CO): d (ppm) 145.8, 144.4 (C-1, C-2),
132.8 (C-4), 120.7 (C-5), 116.5 (C-6), 116.0 (C-3), 40.5 (CH2Ar),
26.7 (CH2S); IR mmax 3466, 3347, 3132, 3030, 2962, 2928, 2904,
2850, 2568, 2360, 1658, 1609, 1517, 1438, 1352, 1273, 1250,
1187, 1134, 1104, 958 cmÀ1; EIMS m/z 170 ([M]+, 37%); HREIMS
m/z calcd for [M]+ C8H10O2S: 170.0402, found: 170.0396.
CO2. After 24 h of incubation, aliquots (100–200 ll) were with-
drawn from the cell suspensions and either used to measure apop-
tosis or centrifuged to recover the supernatant for the subsequent
quantification of H2O2. The proliferation of HL60 and HL60R cells
was determined by counting the viable cells using the trypan blue
exclusion method. Cells were seeded at a density of 0.2 Â 106/mL
in 24-well plates and incubated in the presence of different con-
centrations of phenolic compounds at 37 °C and 5% CO2. After
96 h of incubation 20 lL aliquots were withdrawn from the cell
suspensions, diluted 1:10 with a trypan blue solution (2.22 g/L of
PBS), and, after standing at room temperature for 5 min, the viable
cells were counted using a Burker chamber.
2.12. Apoptosis and cell cycle assays
The percentage of apoptosis was determined by using both fluo-
rescence microscopy and flow cytometry (Hoorens et al., 1996). In
the first case, the cell pellets were resuspended in complete RPMI
medium containing the DNA binding dyes Hoechst 33342 (HO 342,
2.9. Bis(3,4-dihydroxyphenethyl) disulfide (9)
20 lg/ml in PBS) and propidium iodide (PI, 10 lg/ml in PBS). After
To a solution of 6 (100 mg, 0.34 mmol) in EtOH (2 mL), aqueous
2 N HCl (1 mL) was added and the reaction mixture was stirred un-
der argon atmosphere and in darkness at reflux for 24 h. Then, the
reaction mixture was neutralized to pH 5 with a saturated aqueous
solution of NaHCO3 and the solvent was removed under vacuum.
The residue was purified by column chromatography (CH2Cl2–
MeOH 20:1) to give 9 as a reddish oil. Yield 48 mg, 42%; RF 0.37
(CH2Cl2–MeOH 10:1); 1H-RMN (300 MHz, (CD3OD): d (ppm) 6.68
(d, 2H, J5,6 = 8.0 Hz, H-6), 6.64 (d, 2H, J3,5 = 2.0 Hz, H-3), 6.51 (dd,
2H, H-5), 4.93 (br. s, 4H, 4OH), 2.84 (m, 8H, 2CH2S, 2CH2Ar); 13C-
RMN (75.5 MHz, (CD3OD): d (ppm) 146.2, 144.7 (C-1, C-2), 133.2
(C-4), 120.9 (C-5), 116.7 (C-6), 116.4 (C-3), 41.6, 36.1 (CH2Ar,
CH2S); IR mmax 3335, 2924, 2850, 2375, 2346, 1715, 1653, 1605,
1517, 1444, 1366, 1279, 1250, 1192, 1114, 1051 cmÀ1; EIMS m/z
338 ([M]+, 16%); HREIMS m/z calcd for [M]+ C16H18O4S2:
338.0647, found: 338.0651.
10 min of incubation at room temperature, the cells were exam-
ined by using a fluorescent microscope (Zeiss, R.G., equipped with
a 50 W mercury lamp) with ultraviolet excitation at 340–380 nm.
HO 342 is a plasma membrane – permeable compound, which
freely enters the cells with intact membranes, as well as cells with
damaged membranes, and stains the DNA blue, whereas PI is a
highly polar dye that is impermeable to cells with intact plasma
membrane and stains the DNA red. Consequently, the viable cells
have been identified by their intact nuclei with blue fluorescence
(HO 342), and the necrotic cells by their intact nuclei with yellow
fluorescence (HO 342 plus PI). Apoptotic cells were detected by
their fragmented nuclei which exhibited either a blue (HO 342)
or yellow (HO 342 plus PI) fluorescence depending on the stage
of the process. Under each of the experimental condition, three
slides were prepared and 100 cells were counted for each slide.
For the flow cytometry detection of both apoptosis (sub G1) and
cell cycle, the cells were stained with propidium iodide (PI) as fol-
lows: the cell pellet was resuspended in 0.5 mL of hypotonic fluo-
2.10. Cell culture conditions
rochrome solution containing 50 lg/mL of PI in 0.1% sodium citrate
RPMI 1640 and heat-inactivated foetal calf serum (FCS) were
obtained from Gibco (Gibco BRL, Life Technologies, Paisley,
plus 0.1% Triton X-100 in 12 Â 75 mm polypropylene tubes (Becton
and Dickinson, Lincoln Park, NJ, USA). The tubes were kept at 4 °C

M.V. Sepporta et al. / European Journal of Pharmaceutical Sciences 48 (2013) 790–798
793
for 30 min and then the PI fluorescence of individual nuclei was
measured using a FACScan flow cytometer (Becton Dickinson,
Mountain View, CA, USA) at a wavelength of 488 nm. The percent-
ages of cells in G0/G1, S and G2/M phases were calculated using
CellFIT Cell-Cycle Analysis Version 2.0.2. Software.
The regioselective deacylation of ester groups of 6 to afford
derivatives 7–9 was performed by acid hydrolysis (Scheme 2).
The treatment of a solution of 6 in MeOH with aqueous 2 N HCl
(3 equiv.) at r.t. for 12 h gave access to the O-deprotected thioace-
tate 7 in high yield (86%). When heating the reaction mixture at
40 °C for 34 h, complete deacylation of 6 was observed, allowing
the isolation of fully deacetylated compound 4-(2-sulfanyleth-
yl)benzene-1,2-diol (8) in excellent yield (98%). Nevertheless,
when the reaction was carried out under stronger conditions, in
refluxing EtOH for 24 h, the outcome of the reaction was disulfide
9 due to oxidation of the sulfanyl group under the reaction condi-
tions. This disulfide is a particularly interesting target molecule
and could potentially enhance hydroxytyrosol pro-apoptotic and
anti-proliferative activities, since it contains not only the disulfur
functionality involved in redox process in cells, but also two cate-
chol moieties.
2.13. Measurement of H2O2 concentration in the culture medium
The concentration of H2O2 in the culture medium was measured
by the ferrous ion oxidation-xylenol orange method (Nourooz-Za-
deh et al., 1994) as follows: medium (20
tion solution (200 l) containing 250
sulfate, 25 mM H2SO4, 100 mM sorbitol and 125
l
lM
l) was mixed with a reac-
ammonium iron(II)
M xylenol or-
l
l
ange and incubated at room temperature for 30 min. The absor-
bance was then read at 595 nm and the concentration of H2O2
was derived from a standard curve obtained by adding different
concentrations of H2O2 in the RPMI medium just before the assay.
3.2. Pharmacology
2.14. Statistical analysis
The effect of thio derivatives of hydroxytyrosol on apoptosis
and necrosis on both HL60 and HL60R cells was measured by using
fluorescence microscopy after 24 h of treatment (Fig. 1). In the case
of thioacetate 7 and thiohydroxytyrosol 8 it was evident a dose
dependent pro-apoptotic effect while for disulfide 9 a maximum
effect was already reached at 50 lM on both cell lines (Fig. 1A).
In addition, thioacetate 7 and thiol 8 induced very little necrosis
on HL60 cells, whereas disulfide 9 induced significant necrosis on
the HL60 cells particularly at 100 lM. On the HL60R cells a small
All tests were run in triplicate for each experimental condition
and each experiment was repeated at least three times; the results
are reported as the mean SD. Significant differences among treat-
ments were assessed using both Student’s t-test and one-way AN-
OVA. When a significant (p < 0.05) treatment effect was detected,
the mean values were compared using Tukey’s post hoc
comparisons.
but statistically significant necrotic effect was observed for all
3. Results
compounds (Fig. 1B).
Since disulfide 9 resulted the most effective pro-apoptotic com-
3.1. Chemistry
pound at 50 lM, further experiments were performed to test its
pro-apoptotic activity in a lower concentration range (Fig. 2A)
The synthesis of the organosulfur compounds has been carried
out from 3,4-DHPEA 1 via the halogenated analogues 2 and 3
(Scheme 1). The chloroderivative 2 was prepared using a modified
version of Downie’s conditions (Downie et al., 1966), by the treat-
ment of 1 with PPh3 and CCl4 in CH3CN. The iododerivative ana-
and to compare its effect with that induced by 3,4-DHPEA
(Fig. 2B). The data demonstrate that disulfide 9 at 25 lM was about
6 times more effective than 3,4-DHPEA to induce apoptosis in HL60
cells, and about 3 times in HL60R cells. In fact, the AC50% values
(concentration of compound necessary to cause apoptosis on 50%
logue
3
was prepared following
a
modification of Garegg’s
of cells) resulted 15 and 32
>200 M for 3,4-DHPEA on HL60 and HL60R, respectively (Fig. 2).
These results were further supported by the flow cytometry anal-
ysis of sub-G1 peak after treatment of the cells with 25 M of both
compounds (Fig. 3). It is evident that only disulfide 9 was able to
increase the percentage of sub-G1 on both HL60 (Fig. 3A) and
HL60R cells (Fig. 3B) while 3,4-DHPEA did not cause a significant
loss of DNA compared to the control. The PI staining of DNA was
used to investigate the cell cycle distribution of the viable cells
after treatment with the two compounds (Fig. 4). Also in this case
lM for disulfide 9, and 67 and
method (Garegg and Samuelsson, 1980) by the reaction of 1 with
a mixture of PPh3, I2 and imidazole in a ratio of 1:1.5:2, which
showed to be critical for an optimal conversion. Acetylation of
the catechol moiety of both chloro and iodo analogues 2 and 3
led to the di-O-acetyl derivatives 4 and 5 in good yields, which
were employed as starting material to obtain the target organosul-
fur derivatives.
The synthesis of thioacetate 6 was carried out by reaction of
compounds 4 or 5 with potassium thioacetate in refluxing buta-
none (Scheme 1), the reaction time being shorter (2 h) and the
yield higher (76%) when starting from 5, since iodide is a better
leaving group than chloride in a nucleofilic substitution reaction.
l
l
3,4-DHPEA at 25 lM did not significantly modify the phases profile
of both cell lines with respect to the control, while disulfide 9
caused an evident reduction of the number of cells in the G0/G1
Scheme 1.

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M.V. Sepporta et al. / European Journal of Pharmaceutical Sciences 48 (2013) 790–798
Scheme 2.
Fig. 1. Effect of hydroxytyrosol derivatives on apoptosis (A) and necrosis (B) of HL60 and HL60R cells. The cells were incubated in RPMI medium with different compounds for
24 h and then the percentage of apoptotic and necrotic cells were determined by fluorescence microscopy. Values are the mean SD, n = 5; ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001,
compared to the control.
phase with a concomitant increase of cells in the G2/M phase
(Fig. 4). This last effect was particularly evident in the HL60R cells
which showed also a drastic reduction in the number of cells in the
S phase (Fig. 4B). In contrast, disulfide 9 caused on HL60 cells an
increase of the early S phase (Fig. 4A).
Accordingly with these results disulfide 9 resulted more potent
than the other sulfur compounds to reduce the proliferation of
both HL60 (Fig. 5A) and HL60R cells (Fig. 5B) after 96 h of treat-
ment. Worthy of note is the observation that both cell lines show
a similar sensitivity to the anti-proliferative effect of the different
compounds, a very different situation from that observed with
daunorubicin treatment (Fig. 5C). In this case we found that the
HL60 cells were twenty times more sensitive than HL60R to
daunorubicin, in fact the IC50% values (the concentration of dauno-
rubicin that caused the 50% inhibition of growth compared with
control) were 0.0045
lg/ml and 0.1 lg/ml for the HL60 and
HL60R cells, respectively.
Previous data showed that the pro-apoptotic ability of 3,4-
DHPEA was mostly due to the extracellular production of H2O2
(Fabiani et al., 2009). We wondered whether the same phenome-
non occurs with the thio derivatives of hydroxytyrosol. The cell
death caused by both 3,4-DHPEA 1 and the hydroxytyrosol deriva-
tives 7–9 (100 lM) was measured in the absence and in the pres-
ence of catalase (CAT: 100 U/ml) (Table 1), an enzyme that
converts hydrogen peroxide to water and oxygen. Furthermore,
the accumulation of H2O2 in the culture medium was also

M.V. Sepporta et al. / European Journal of Pharmaceutical Sciences 48 (2013) 790–798
795
determined after incubation of the cells for 24 h with the different
compounds (Table 1). Regarding HL60 cells, as expected, 3,4-
DHPEA caused an increase of H2O2 in the culture medium and its
pro-apoptotic activity was inhibited by CAT (Table 1). On the con-
trary, the enzyme did not significantly reduce apoptosis induced by
all thio derivatives, even if, in the case of disulfide, there was a sig-
nificant accumulation of H2O2 in the culture medium (Table 1).
When the HL60R cells were considered, 3,4-DHPEA induced a
low value of apoptosis while the thio derivatives were more active
with a pro-apoptotic effect statistically significant compared to the
control. In addition, treatment with disulfide 9 induced also a sig-
nificant necrotic effect on the HL60R cells. These results are of par-
ticular relevance since the HL60R cells are resistant to the
treatment with the most common chemotherapeutic agents such
us daunorubicin and cytosine arabinoside (Table 1). Finally, the
accumulation of H2O2 in the culture medium after treatment with
3,4-DHPEA 1 and disulfide 9 was clearly lower in the presence of
HL60R cells when compared to HL60, so suggesting that the former
are more active in removing the H2O2 from the culture medium.
4. Discussion
Several evidences have demonstrated that natural products
continue to play a significant role in the drug discovery and devel-
opment process. Indeed, in the case of cancer treatments, over the
period of time between early 1940s to date, of the 175 small mol-
ecules approved as therapeutic agents about 50% are either natural
products or compounds directly derived therefrom (Newman and
Cragg, 2012). The main goal of the strategies for anticancer drugs
development is to find out molecules able to inhibit the prolifera-
tion and/or to kill tumor cells. Cell death is generally discussed in
term of either apoptosis or necrosis. Apoptosis is an active,
Fig. 2. Effect of increasing concentration of disulfide 9 (A) and 3,4-DHPEA (B) on
apoptosis of HL60 and HL60R cells. The cells were incubated in RPMI medium with
increasing concentrations of compounds for 24 h and then the percentage of
apoptotic and necrotic cells were determined by fluorescence microscopy. Values
are the mean SD, n = 5. Means without a common letter differ, p < 0.05.
Fig. 3. Effect of 3,4-DHPEA and disulfide 9 on the sub G1 peak of HL60 (A) and HL60R (B) cells. The cells were incubated in RPMI medium with 25 lM of the two compounds
for 24 h and then the percentage of apoptotic cells (sub G1) were determined by PI staining and flow cytometry. It is shown a representative experiment of three performed
which gave similar results.

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M.V. Sepporta et al. / European Journal of Pharmaceutical Sciences 48 (2013) 790–798
Fig. 4. Effect of 3,4-DHPEA and disulfide 9 on the cell cycle distribution phases of HL60 (A) and HL60R (B) cells. The cells were incubated in RPMI medium with 25 lM of the
two compounds for 24 h and then the percentage of cells in the different phases of cycle were determined by PI staining and flow cytometry. It is shown a representative
experiment of three performed which gave similar results.
regulated and programmed process characterized by chromatin
condensation, membrane blebbing, cell shrinkage and DNA frag-
mentation while necrosis has been characterized as passive and
accidental cell death, resulting from environmental perturbations
followed by uncontrolled release of inflammatory cellular contents
(Fink and Cookson, 2005). In this study we have synthesized thio
derivatives of hydroxytyrosol possessing both catechol moiety
and sulfur containing functions on a single molecule, and com-
pared their anti-proliferative, pro-apoptotic and necrotic activities
on promyelocytic HL60 and HL60R cells. The main findings were as
follow: (i) all thio derivatives of hydroxytyrosol were more effec-
tive than 3,4-DHPEA in inducing apoptosis on HL60R cells; (ii)
hydroxytyrosol disulfide 9 was the most active anti-proliferative
and pro-apoptotic compound on both HL60 and HL60R cells; (iii)
different from 3,4-DHPEA, all thio derivatives of hydroxytyrosol in-
duced apoptosis by a mechanism that did not involve the release of
H2O2 in the culture medium; (iv) the straightforward access to the
thio derivatives from 3,4-DHPEA through high-yielding pathways
and the strong activity shown make them candidates for further
studies in cancer therapy.
Many studies have clearly shown that reactive oxygen species
(ROS) may act as signaling molecules for the initiation and the exe-
cution of apoptosis (Ozben, 2007). This mechanism has been dem-
onstrated to participate in the 3,4-DHPEA induced apoptosis on
HL60 cells, which are particularly sensitive to the effect of H2O2
(Fabiani et al., 2012) whereas it seems not to be involved in the
case of thio derivatives of hydroxytyrosol. In particular, the results
obtained with disulfide 9 are remarkable in this context because
this compound was more active than 3,4-DHPEA (it induced apop-
tosis in about 75% of the HL60 cells at a 4 times less concentration
in comparison to 3,4-DHPEA) and although able to release H2O2 in
the culture medium, this property was not involved in its pro-
apoptotic activity. On the other hand, thioacetate 7 did not induce
a significant accumulation of H2O2 in the culture medium. The
apparent less accumulation of H2O2 induced by the thioacetate
derivative with respect to hydroxytyrosol or disulfide 9 could be
explained by the easier oxidation of the thioacetyl function.
Among the different classes of natural organosulfur compounds,
those containing a core disulfide structure have been deeply inves-
tigated. Most of these studies were focused on natural disulfides
abundantly and mainly present in allium vegetables, in particular
on diallyl disulfide (DADS). Differently from our data on hydroxy-
tyrosol disulfide 9, it was reported that DADS induced apoptosis
on HL60 cell throw the generation of hydrogen peroxide since both
NAC and CAT were able to prevent this effect (Kwon et al., 2002).
Furthermore, worthy of note is also the observation that hydroxy-
tyrosol disulfide 9 resulted more potent to induce apoptosis com-
pared to DADS, indeed we found that at 25 lM hydroxytyrosol
disulfide 9 induced apoptosis on over 70% of HL60 cells while at
the same concentration DADS was effective only on 17% HL60 cells
(Kwon et al., 2002). These results suggest that the molecular struc-
ture of regions flanking the disulfide group may deeply influence
both the potential and the mechanisms by which these compounds
induce tumor cell death. In agreement with this observation a
recent study has described the structure–activity relationships
between different synthesized organosulfur compounds with their
ability to induce apoptosis on human leukemia cells (Wong et al.,
2010).
The mechanisms by which these new thio derivatives of
hydroxytyrosol induce apoptosis on HL60 are currently unknown.
In the case of DADS apoptosis was associated to the activation of
caspase-3 and degradation of PARP (Kwon et al., 2002). In addition,
the DADS induced-apoptosis on HL60 cells was related to a signif-
icant antiproliferative effect and to a G2/M phase arrest of the cell
cycle (Yi et al., 2009). Similarly to DADS we found that disulfide 9
caused an increase of G2/M phase on both HL60 and HL60R cells.
Curiously, an opposite effect of disulfide 9 was observed on the S
phase which was increased and reduced in HL60 and HL60R cells,

M.V. Sepporta et al. / European Journal of Pharmaceutical Sciences 48 (2013) 790–798
797
it was found that DADS is able to induce differentiation on HL60
cells through inhibition of JAK/STAT (Wu et al., 2005, 2003).
Whether similar molecular mechanisms and differentiating effect
are induced by hydroxytyrosol disulfide 9 on HL60 cell remain to
be determined. It is possible that thiol-disulfide exchange mecha-
nisms between hydroxytyrosol disulfide 9 and some cysteine resi-
dues of different important proteins might be involved.
The observation that the thio derivatives of hydroxytyrosol
were more potent inducers of cell death on HL60R with MDR phe-
notype than both hydroxytyrosol and some anti-cancer drugs is of
particular relevance. Although the phenomenon of resistance to
anti-cancer drugs appears to be multi-factorial involving different
mechanisms like inactivation of drugs and alteration of targets in-
volved in the DNA repair and apoptosis, the most important feature
seems to be the overexpression of transmembrane proteins
belonging to the family of ATP Binding Cassette Transporters
(ABC), in particular P-glycoprotein GP170 (Ambudkar et al., 1999;
Stavrovskaya, 2000). These proteins mediate the efflux of different
compounds across the plasma membrane in both physiological and
pathological conditions. Since their overexpression has been asso-
ciated with both the fatal outcome of cancer and the failure of can-
cer therapies many studies have been devoted to find out specific
inhibitors. Actually there are available different ABC inhibitors like
verapamil and nifelpidin but the main problem with their use is
that they are selective against a specific drug and often they have
a high toxicity (Ullah, 2008). Therefore, the discovery of natural
molecules or synthetic derivatives able to modulate the MDR phe-
nomenon without detrimental effects would be of particular
importance. Indeed, recent studies have demonstrated that some
natural compounds such us flavonoids are able to modulate the
MDR phenotype by the interaction with both the ATP binding site
and the hydrophobic portion of P-glycoprotein (Chung et al., 2005;
Conseil et al., 1998). Further examples of natural compounds that
have shown an inhibitory effect on P-glycoprotein with the conse-
quent reversal of the multidrug resistance are (–)-epigallocatechin
gallate and curcumin (Feng et al., 2005; Choi et al., 2008). Although
our data clearly indicate that the thioderivatives of hydroxytyrosol
are able to induce apoptosis on HL60R, further studies are neces-
sary to clarify their mechanisms of action and in particular their
modulatory effect on the P-glycoprotein activity.
Fig. 5. Effect of 3,4-DHPEA and its derivatives on the proliferation of HL60 (A) and
HL60R (B) cells. For comparison in (C) it is shown the antiproliferative effect of
daunorubicin on both cell type. The cells were incubated in RPMI medium with
increasing concentrations of the different compounds for 96 h and then the number
of viable cells were determined by trypan blue exclusion test. The results are
expressed as % of control. Values are the mean SD, n = 5.
5. Conclusion
respectively. DADS exerted also inhibition of ERK, activation of p38
and JNK signaling pathway, downregulation of Bag-1 and Bcl-2,
and upregulation of Fas-L and Bax (Yi et al., 2009). Furthermore,
We have shown that the introduction of sulfur containing func-
tional groups on hydroxytyrosol molecule leads to an enhancement
Table 1
Effect of catalase (CAT) on apoptosis, necrosis and H2O2 accumulation induced by different compounds at 100 lM.
Treatments
HL60
HL60R
Apoptosis (%)
Necrosis (%)
H2O2
lM
Apoptosis (%)
Necrosis (%)
H2O2 lM
None
5.8 1.2d
87.5 6.4a
27.3 8.9d
75.8 4.8a,b,c
65.4 27.2b,c
90.1 1.9a
0.6 0.5c
5.7 0.3c
1.5 0.5c
1.5 1.5c
3.8 2.8c
2.2 1.9c
8.3 8.2c
36.0 10.5b
29.5 2.6b
92.8 5.3a
0.3 0.2c
0.4 0.1b
4.2 0.6a
0.9 0.4b
1.2 0.4b
0.5 0.1b
6.8 1.1e,f
24.4 4.8d,e
20.0 5.4e,f
46.8 12.4c
41.8 11.4c,d
55.1 14.4b,c
62.5 9.1a,b
67.5 9.7a
2.3 1.3d
16.4 8.4b,c,d
6.1 5.6d
0.4 01c
1.6 0.3a
1.0 0.8a,b,c
0.4 0.1c
0.4 0.1c
N.D.
3,4-DHPEA 1
1 + CAT*
Thioacetate 7
7 + CAT
Thiohydroxytyrosol 8
8 + CAT
8.3 3.5c,d
5.2 2.5d
N.D.§
13.2 9.6b,c,d
10.2 9.0c,d
21.3 2.9b,c
26.6 6.3b
48.8 11.9a
4.8 3.9d
86.9 9.2a,b
63.2 10.4c
72.0 1.0a,b,c
7.2 5.3d
N.D.
N.D.
Disulfide 9
9 + CAT
4.7 1.0a
0.5 0.1b
N.D.
1.2 0.1a,b
0.7 0.1b,c
N.D.
60.3 11.1a,b,c
3.2 2.9f
DNR**
AraC***
71.5 6.1a,b,c
N.D.
19.6 8.7e,f
N.D.
Means in a column without a common letter differ, Tukey’s post hoc comparisons p < 0.05.
*
CAT: catalase was included in the culture medium at 100 U/ml.
**
DNR: daunorubicin 2
lM.
***
AraC: cytosine arabinoside 2
lM.
§
N.D.: not determined.

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M.V. Sepporta et al. / European Journal of Pharmaceutical Sciences 48 (2013) 790–798
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l
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Appendix A. Supplementary material
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