Effect of Allyl Sulfides from Garlic Essential Oil on Intracellular Ca
2+
Levels in Renal Tubular Cells
Chung-Ren Jan,
†
Horng-Ren Lo,
‡
Chung-Yi Chen,
‡
and Soong-Yu Kuo*
,‡
†
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan
‡
Department of Medical Laboratory Science and Biotechnology, School of Medical and Health Sciences, Fooyin University,
Kaohsiung 83102, Taiwan
ABSTRACT: Diallyl sulfide (1), diallyl disulfide (2), and
diallyl trisulfide (3), which are major organosulfur compounds
of garlic (Allium sativum), are recognized as a group of
potential chemopreventive compounds. In this study, the early
signaling effects of 3 were examined on Madin-Darby canine kidney (MDCK) cells loaded with the Ca
2+
-sensitive dye fura-2. It
was found that 3 caused an immediate and sustained increase of [Ca
2+
]
i
in a concentration-dependent manner (EC
50
= 40 μM).
Compound 3 also induced a [Ca
2+
]
i
elevation when extracellular Ca
2+
was removed, but the magnitude was reduced by 45%. In
Ca
2+
-free medium, the 3-induced [Ca
2+
]
i
level was abolished by depleting stored Ca
2+
with 1 μM thapsigargin (an endoplasmic
reticulum Ca
2+
pump inhibitor). Elevation of [Ca
2+
]
i
caused by 3 in the Ca
2+
-containing medium was not affected by modulation
of protein kinase C activity. The 3-induced Ca
2+
influx was inhibited by nifedipine and nicardipine (1 μM). U73122, an inhibitor
of phospholipase C, abolished ATP (but not the 3-induced [Ca
2+
]
i
level). These findings suggest that 3 induced a significant
[Ca
2+
]
i
elevation in MDCK renal tubular cells by stimulating both extracellular Ca
2+
influx and thapsigargin-sensitive intracellular
Ca
2+
release via as yet unidentified mechanisms. Furthermore, the order of the allyl sulfide-induced [Ca
2+
]
i
elevation and cell
viability was 1 < 2 < 3. The differential effect of allyl sulfides on Ca
2+
signaling and cell death appears to correlate with the
number of sulfur atoms in the structure of these allyl sulfides.
S
everal population-based studies show that inhabitants of
Southeast Asian countries have a much lower risk of colon,
gastrointestinal, prostate, breast, and other cancers than their
European and American counterparts.
1
It is very likely that
constituents of their diet such as garlic, ginger, and chillies may
play important roles in cancer prevention. Garlic (Allium
sativum L.; Alliaceae) has been used widely as a flavoring agent
in cooking and as a medicinal herb in traditional medicine.
2
Garlic-derived organosulfur compounds have been reported to
reduce chemically induced colon, esophageal, lung, mammary,
pulmonary, skin, and stomach tumors.
3-7
Among these sulfur-
containing compounds, diallyl sulfide (1), diallyl disulfide (2),
and diallyl trisulfide (3) are the three major components in
garlic volatile oil. The potential chemopreventive effects of allyl
sulfides have been attributed not only to the modulation of the
antioxidative and/or drug-metabolizing enzyme systems, but
also to the inhibition of cell proliferation and induction of
apoptosis for tumor cells.
8
Various pathways of these essential garlic-derived allyl
sulfides have been suggested for anticarcinogenic activity,
including modulation of xenobiotic-metabolizing enzyme
activities,
9
inhibition of DNA adduct formation,
10
modulation
of signal transduction pathways,
11
reduction of proliferation,
12
and induction of apoptosis.
12
Recently, molecular changes of
allyl sulfides in G2/M arrest,
12,13
β-tubulin oxidation,
14
and
increase of cytosolic Ca
2+
([Ca
2+
]
i
) were shown.
15,16
In
addition, the possible role of Ca
2+
in apoptosis induced by
diallyl disulfide (2) was described in colon cancer cells and
retinal ganglion cells.
17,18
It has been shown that BAPTA (an
intracellular Ca
2+
chelator) can suppress 2-evoked [Ca
2+
]
i
elevation, and that reactive oxygen species (ROS) generation
can prevent caspase 3 activation and apoptosis. However,
despite the accumulation of data, the underlying molecular
mechanism of Ca
2+
signaling by diallyl trisulfide (3) is still
unclear.
It is known that Ca
2+
ions serve as a ubiquitous second
messenger in all eukaryotic cells.
19
Under physiological
conditions, the resting [Ca
2+
]
i
is maintained at levels less
than 0.1 μM, about four orders of magnitude lower than in the
extracellular environment (1-2 mM), but cellular excitation
induces a transient [Ca
2+
]
i
elevation up to several mM, or to
even higher levels in tiny cellular compartments. These
transient fluctuations of [Ca
2+
]
i
(termed the “Ca
2+
signal”)
trigger or regulate various intracellular events. It is well
established that cellular Ca
2+
overload, or perturbation of
intracellular [Ca
2+
]
i
levels, may cause cytotoxicity and result in
either apoptosis, necrosis, or autophagy. In general, the
generation of Ca
2+
signal is determined by interaction of (1)
external Ca
2+
entry, (2) Ca
2+
release from intracellular
compartments (Ca
2+
stores), (3) cytoplasmic Ca
2+
buffering
by Ca
2+
binding proteins, and (4) subsequent Ca
2+
removal
from the cytoplasm due to transmembrane Ca
2+
efflux or
sequestration by intracellular Ca
2+
stores located in the
organelles.
20
The aim of the present study was to explore the effect of the
allyl sulfides 1-3 on [Ca
2+
]
i
in renal tubular cells. Madin-Darby
Received: July 30, 2012
Published: November 19, 2012
Article
pubs.acs.org/jnp
© 2012 American Chemical Society and
American Society of Pharmacognosy 2101 dx.doi.org/10.1021/np3005248 | J. Nat. Prod. 2012, 75, 2101-2107