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disaccharide 5 was monitored by the oxidation-sensitive fluores-
HL-60
U937
cent dye 20,70-dichlorodihydrofluorescein diacetate (H2-DCF-DA).
The results (Fig. 5) demonstrate that treatment of HL-60 cells with
5 for up to 12 h led to a decrease in the levels of ROS.
46 kDa
Pro-caspase 9
Pro-caspase 7
35-37 kDa
While there are effective treatments for acute lymphocytic
leukemia and for chronic myelogenous leukemia, more effective
treatments for other forms of acute leukemia are needed. In this
35 kDa
32 kDa
study we have evaluated the effect of twelve alkyl
a/b-(1?6)-
diglucopyranosides on cell viability of four human tumor cell lines.
Interestingly, we found that four of these compounds display cyto-
toxic properties in human leukemia (HL-60, U937 and Molt-3) and
in breast cancer (MCF-7) cells. In general, all compounds displayed
similar potency in all human tumor cells evaluated in this study.
Pro-caspase 6
Pro-caspase 3
34 kDa
36 kDa
Inhibition of cell viability by menthyl
a/b-(1?6)-diglucopyrano-
sides was accompanied by an increase in the number of apoptotic
cells as determined by fluorescent microscopy and also by flow
cytometry. Our results also indicate that (+)-menthyl b-(1?6)-
diglucopyranoside 5 displays an antiproliferative effect which is
dependent on caspase, since cell death was inhibited by the gen-
eral caspase inhibitor z-VAD-fmk.
18-20 kDa
116 kDa
85 kDa
PARP
Fragment
The intrinsic apoptotic pathway (mitochondrial pathway) in-
volves the release of cytochrome c and the assembly of apoptosome.
15 kDa
26 kDa
Cyt-c
Bcl-2
Bax
We also observed
a concentration-dependent processing of
caspase-9, in accordance with the cytochrome c release experi-
ments, emphasizing that the intrinsic pathway plays an important
role in 5-induced cell death. Moreover, disaccharide 5 induced PARP
[poly(ADP-ribose) polymerase] cleavage, a hallmark of apoptosis
that indicates activation of caspase-3 and -7. Although PARP is also
degraded in other forms of cell death like necrosis,9 this did not
seem to be the case with 5 in accordance with the fluorescence
microscopy experiments and also by the generated PARP fragments.
The results demonstrate that compound 5 also stimulates the prote-
olytic processing of other executioner caspases, namely caspase-7
and caspase-6 to form activated enzymes and contributing in the
process of cell death triggered by this menthyl diglucopyranoside.
There was also an increase in the cytosol Bax levels in both HL-60
and U937 cell lines. Although the activation of Bax during apoptosis
usually does not require an increase in genetic transcription, various
chemotherapeutic treatments aim at up-regulation of the Bax gene
to block tumor progression. For example, all the antracycline-group
chemotherapeutic agents fight cancer through enhancement of Bax
expression.10 Moreover, the results clearly demonstrate that the
(+)-menthyl b-(1?6)-diglucopyranoside 5 induces Bid cleavage,
since the levels of Bid were clearly reduced in accordance with the
initiator caspase-8 activation.
22 kDa
22 kDa
42 kDa
Bid
β-Actin
0 10 30
0 10 30
5 (µM)
Figure 3. Immunoblotting for the cleavage of caspases and PARP, cytochrome c
release and Bcl-2 family members after 24 h incubation with the selected (+)-
menthyl b-(1?6)-diglucopyranoside 5 on human leukemia cells. b-Actin was used
as a loading control.
The reactive oxygen species are toxic byproducts of aerobic
metabolism and play significant roles in different signal transduc-
tion pathways, including those leading to apoptosis.11 In this study
we show that 5 decreased the ROS levels. Although the mecha-
nisms by which this compound caused this response is unknown,
one possible explanation could be the partial retention of cyto-
chrome c within the mitochondria which could catalyze the oxida-
tion of superoxide to oxygen,12 or perhaps this (+)-menthyl b-
(1?6)-diglucopyranoside might increase the level of antioxidants
such as reduced glutathione or superoxide dismutase.
lysates were assayed for cleavage of the tetrapeptide DEVD-pNA,
IETD-pNA and LEHD-pNA as specific substrates for caspase-3/7, cas-
pase-8 and caspase-9, respectively. As shown in Figure 4A, the re-
sults demonstrate a >7-fold increase in caspase-3/7 activity in all
leukemia cell lines, whereas caspase-9 and caspase-8 showed a les-
ser degree of activation, consistent with their role as initiator
caspases.
To confirm that caspases are involved in disaccharide 5-induced
apoptosis, HL-60 cells were pre-treated with the broad-spectrum
caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe) fluorom-
ethyl ketone (z-VAD-fmk). The results (Figure 4B and C) show that
apoptosis was completely suppressed in the presence of the inhib-
itor, which suggests that the (+)-menthyl derivative 5 induces
cytotoxicity by a caspase dependent mechanism.
In conclusion, the selected synthetic menthyl
a/b-(1?6)-
diglucopyranosides evaluated in the present study are strongly
cytotoxic against human leukemia cell lines, and in the case of
5, it involves
a caspase dependent apoptosis and operates
through a mechanism independent of ROS generation. The chem-
ical synthesis of this kind of compounds might allow the discov-
ery of new and highly specific antitumor agents against leukemia
cells.
Intracellular reactive oxygen species (ROS) are considered one
of the key mediators of apoptotic signaling for many antitumoral
agents. Therefore, the redox status of HL-60 cells treated with