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Introduction
The development of agents that induce differentiation and/or apoptosis is a promising avenue for the treatment of human
acute myeloid leukemia HL-60 cells. Epidemiologic studies offer evidence that a high garlic consumption reduces the risk of
colorectal and stomach cancers[1]. These observations are supported by
in vivo experiments carried out in rodents,
concluding that garlic extract or garlic powder intake reduces chemically-induced carcinogenesis in different organs (the skin and the
mammary gland)[2_4]. Diallyl disulfide (DADS) is a naturally occurring organosulfur compound derived from crushed garlic,
which is the predominant lipid-soluble sulfide in essential garlic
oil[5]. Some studies have shown that the antiproliferative
activity of DADS is believed to be due to its ability to induce differentiation
and/or apoptosis and to arrest cells in the
G0/G1 or G2/M
phase[6_9] in A549 human lung cancer, T24 human bladder cancer, breast, colon cancer cells,
etc[10_13]. DADS-induced apopto-sis in human leukemia HL-60 cells is triggered by the generation of hydrogen peroxide, activation of caspase-3,
degradation of poly(ADP-ribose) polymerase (PARP), and fragmentation of
DNA[14]. In addition to the antiproliferative
activity in cell culture and tumor xenograft models, DADS is highly effective for the prevention of chemically-induced
cancers in animal models. Wattenberg et al were the first to demonstrate that
N-nitrosodiethylamine-induced neoplasia of the
forestomach in female A/J mice was inhibited by 90% upon
po DADS administration prior to the carcinogen
challenge[15]. The intragastric intubation of DADS also prevented colon and renal neoplasia in the multi-organ carcinogenesis model in male
F344 rats[16].
The acetylation of histones appears to be an important mechanism for the regulation of gene
transcription[17_19]. The acetylation of specific lysine residues, which occurs within the N-terminal domain of core histones, is one of the mechanisms
involved in the modification of the chromatin structure, and is generally correlated with transcriptional gene activity. Histone
acetylase (HAT) and deacetylase (HDAC) are emerging as important components of protein complexes that affect the
dynamics of chromatin folding during gene transcription. In normal cells, histone acetylation levels are thought to result from
an equilibrium between competing HAT and
HDAC[20,21]. Histone acetylation is often associated with activated transcription,
and deacetylation correlates with transcriptional silencing. Recently, some studies have shown that DADS antiproliferation
is related to the increase of histone acetylation. DADS antiproliferative effects in human colon tumor cell lines HT-29 and
Caco-2[22,23], and in DS19 mouse erythroleukemic
cells[24] ,were associated with a transient increase of histone acetylation;
more-over, DADS could inhibit nuclear HDAC activity. We previously reported that DADS could significantly inhibit the
growth of MGC803 cells in vitro and in
vivo, by activating p38 MAPK pathways to induce G2/M arrest of
cells[25,26]. DADS had a significant antiproliferative effect and could also induce differentiation in leukemia HL-60
cells[27_29]. The objective of the present work was to establish whether DADS suppresses proliferation and induces differentiation of HL-60 cells through
increasing histone acetylation and the expression of
p21WAF1 in vitro and in
vivo.
Materials and methods
Reagents DADS (purity 80%, the remaining 20% being diallyl trisulfide and diallyl sulfide), purchased from Fluka
Co (Milwaukee, Wisconsin, USA), was dissolved in
Tween 80[28]at 8 g/L and stored at -20 °C. Sodium butyrate (SB), which was
purchased from Sigma Co (St Louis, Montana, USA), was dissolved in PBS at 100 mmol/L and stored at -20 °C it was used as
the positive control.
Animals The severe combined immunodeficiency (SCID) mice, eighteen 3_4-week-old male (11_12 g), purchased from the
Experimental Center of the Chinese Academy of Science in Shanghai, were fed common mouse feeds with eggs and
maintained under specific pathogen-free conditions in the animal laboratory of our Cancer Research Institute.
Cell culture HL-60 cells, obtained from the Cancer
Research Institute, Xiangya Medical College, Center South University in China, were cultured in RPMI-1640 medium with
10% fetal calf serum at 37 °C in a 5%
CO2 incubator.
Differentiation assays Nitroblue tetrazolium (NBT) reduction was performed by the previously described methods to
measure the differentiation induction of the HL-60
cells[29].
Protein extraction Cultures of HL-60 cells in the logarithmic growth phase at a density of
2×105 cells per mL were cultured with 1.25
µg/mL DADS for 12, 24, 48, or 72 h at 37 °C.
Cells cultured without DADS were harvested for comparison of histone
acetylation levels. At the appropriate time point, the cells were harvested, washed with ice-cold PBS and suspended in 0.5 mL
lysis buffer [10 mmol/L Tris-HCl (pH 7.6), 100
mmol/L NaCl, 1 mmol/L EDTA (pH 8.0) and 100
µg/mL PMSF] containing protease inhibitor aprotinin (1 µg/mL). Lysates were centrifuged at 10
000×g for 10 min, and extracts were quantified using a
background-corrected absorbance (BCA) protein quantified kit (Pierce, Rockford, IL, USA).
Inoculation of SCID mice
HL-60 5×106 cells in the logarithmic growth phase were injected into the right side of the
peritoneal cavity per mouse, and the mice were monitored until the peritoneal neoplasms were detected (28 d after the
inoculation of the HL-60 cells).
Treatment of SCID mice with DADS
SCID mice bearing HL-60 peritoneal neoplasms were randomly divided into 3 groups
(6 mice per group). The mice received an ip injection of vehicle alone (NS), 42 mg/kg DADS or 73 mg/kg SB 3 times per week,
for a total treatment period of 21 d. Injection volumes were kept constant at 0.2 mL for each mouse. Tumor length and width
and abdomen circumference were measured twice weekly by vernier calipers, and tumor volume was calculated using the
following formula:
Tumor
volume=length×(width)2 ×p/6
After 21 d of treatment, at which time the control animals (third and fifth) had large neoplasms which required the animals
to be sacrified, all of the mice were killed 12 h after the final injection and the neoplasms and livers were removed. Tissues
were flash-frozen in liquid nitrogen or fixed in formalin and embedded in paraffin. During the treatment period, the mice were
weighed twice weekly and monitored for any overt signs of toxicity.
Cycle distribution analysis of HL-60 cells in
SCID The cycle distribution of HL-60 cells in SCID was monitored by flow
cytometry analysis as follows: 100 mg tumor tissues were cut into mono-cell suspension in ice-cold PBS. The suspension
cells were enumerated by a counter, and
1×106 cells were pelleted by centrifugation. The supernatant was aspirated and the
cells were resuspended in 1 mL of ice-cold ethanol (75%) and stored for 24 h at 4 °C. After the ethanol was removed, the cells
were incubated with PBS containing RNase at 37 °C for 30 min and then stained for 30 min with propidium iodide. Sample data
were collected using a Becton Dickinson FACS (Franklin Lakes, NJ, USA) and analyzed with Verity Winlist Software (Verity
Software House, Topsham, ME, USA).
Tissue protein
extraction Frozen tumor tissues 100 mg were ground under liquid nitrogen and washed in ice-cold PBS,
the PBS was discarded and the tissues were suspended
in 0.5 mL lysis buffer [10 mmol/L Tris-HCl (pH 7.6), 100
mmol/L NaCl, 1 mmol/L EDTA (pH 8.0) and 100 µg/mL PMSF] containing protease inhibitor aprotinin (1µg/mL). After centrifugation at 10
000×g for 10 min, the supernatant collected was analyzed for protein content using a BCA protein quantified kit (Pierce,
Rockford, IL, USA).
Western blot analysis Total proteins (20_25 µg/point) were separated by 12% SDS-PAGE and transferred to a
polyvinylidene fluoride membrane. The blots were blocked
in 5% nonfat milk in Tris buffered saline (TBS)
containing 0.1% Tween 20 for 2 h at room temperature, and incubated for 2 h at room temperature with the 1:1000 dilution of the acetylated
histones H3 and H4 rabbit polyclonal primary antibodies (Upstate Biotechnology, Lake Placid, NY, USA) and with the 1:400
anti-p21WAF1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blots were washed for 3×5 min in TBS-T and
then incubated with a 1:1000 dilution of peroxidase-conjugated secondary antibody for 1 h at room temperature. The blots
were again washed for 3×10 min in TBS-T and then developed by an enhanced chemiluminescence plus (ECL Plus) kit
(Amersham Biosciences, Buckinghamshire, England). Where indicated, the blots were stripped and reprobed with
antibodies directed against β-actin (Sigma-Aldrich, St Louis, Montana, USA).
Statistical analysis Data were expressed as Mean±SD. Group
t-test was used to compare data between every 2 groups.
One-way analysis of variance (ANOVA) was used to determine statistically significant differences among multiple groups.
P<0.05 was considered statistically significant.
Results
Differentiation induction by DADS in HL-60
cells Table 1 showed the NBT reduction ability of HL-60 cells treated with
DADS increased in a time-dependent manner, compared with no treatment of HL-60 cells
in vitro (P<0.05).
Induction of histone H4 and H3 acetylation in HL-60 cells by
DADS DADS increased the acetylation levels of
histones H4 and H3 in HL-60 cells. The effect of 1.25
µg/mL DADS on histone H4 and H3 acetylation was studied by Western blot.
DADS induced histone H3 and H4 hyper-acetylation after 24 h incubation with significant 2.0-fold and 2.4-fold increase,
respectively (Figure 1).
DADS increases
p21WAF1 protein expression in HL-60
cells The p21WAF1 protein is an essential regulatory protein of cell
cycle progression. Expression of its gene is partly regulated by histone acetylation. Since we observed that
1.25 µg/mL DADS induced histone hyperacetylation and increased HL-60 cell differentiation, we studied its effects on
p21WAF1 protein levels. DADS 1.25
µg/mL induced significant increases in
p21WAF1 protein levels after 24-h incubation (Figure
2).
Growth inhibition of the peritoneal neoplasm by DADS
In order to evaluate the effects of DADS on HL-60 cell growth
in vivo, we examined its antitumor efficacy using a SCID mouse model. After 21 d of treatment, the animals were killed and the
tumor sizes were determined. The mean tumor volume in mice treated with NS was 5.47±0.57
cm3, the mean tumor volume in mice treated with DADS was 1.85±0.30
cm3, and that in the group treated with SB was 1.92±0.30
cm3. The difference in tumor size between the mice treated with DADS and those treated with SB did not achieve statistical significance
(P>0.05), whereas the group treated with DADS showed markedly suppressed tumor growth compared with the NS control
(P<0.01) (Figure 3).
To monitor any possible toxicity arising from the treatment, the mice from all of the treatment groups were weighed twice
weekly during the 21-d treatment period, No difference was detected among bundles and no mouse died throughout the
treatment period.
Cycle distribution of HL-60 cells in
SCID The cycle distribution of HL-60 cells in SCID mice was monitored by flow
cytometry analysis. The percentages of
G0/G1 or S phase cells in the NS group were 25.4% and 61.7%,
respectively. In SB, an
contrast, the 42 mg/kg DADS and 73
mg/kg SB treatment led to a significant inhibition of DNA synthesis as evidenced by
the fact that the percentages of
G0/G1 phase cells increased to 63.4%; the S phase cells decreased to
27.8% in the DADS group (Figure 4).
DADS increases acetylated histones
in vivo Proteins were isolated from the excised tumors of 3 mice in each treatment
group at the end of the 21-d treatment period. The tumors removed from the mice treated with 42 mg/kg DADS showed
significant increase of acetylated histone H3 and H4, 4.1-fold and 3.2-fold increases, respectively, compared with the tumors
from the NS group. There was no difference compared with the tumors of the mice treated with 73 mg/kg
inhibitor of histone deacetylase (Figure 5).
Increase of
p21WAF1 expression in tumor cells by
DADS Protein lysates were isolated from the sample removed from 3
mice in each group and analyzed for expression of the
p21WAF1 protein. The tumors from the mice treated with DADS showed
a 2.8-fold increase in the expression of the
p21WAF1 protein, compared with tumors from the NS group; there was also a
resemblance to the expression in the SB-treated group, an inhibitor of histone deacetylase (Figure 6).
Discussion
This study demonstrates that DADS is a potent inhibitor of HL-60 cell growth
in vitro and in vivo. In
vitro, our previous work showed that 1.25 µg/mL DADS inhibited HL-60 cell proliferation and induced differentiation. The cell surface
differentiation antigen CD11b increased from 12% (untreated) to 44% (treated by 1.25
µg/mL DADS for 3 d) (P<0.01). G0/G1 cells were
added to 44.6% (P<0.01), but S and
G2/M phase cells descended to 48.5% and 6.9%
(P<0.01), respectively (the ratio of
G0/G1, S, and G2/M phase in untreated HL-60 cells were 23%, 66% and 11%, respectively), and the cell cycle was arrested at the
G1 phase. Kinase activity of Janus kinase (JAK)/ signal transduction and activators of transcription (STAT) was tested by
Western blot. The results demonstrated that the activity of phosphorylated Jak1 and Stat3 kinase was inhibited, the
expression of Stat3 and c-myc gene decreased,
c-fos and c-jun gene expression increased in HL-60 cells treated by DADS. The
above results suggest that DADS could induce HL-60 cells differentiation toward granulocytic lineage, the inhibition of
phosphorylated Jak1 and Stat3 was involved in HL-60 cell differentiation induced by DADS, and its possible molecular
mechanism might relate to the modulation of proliferation-associated gene expression and inhibition of DNA
synthesis[28_30]. In the present study, the NBT-reduction assay showed that the percentage of NBT-positive cells markedly increased after
treatment with DADS, which suggests that DADS can induce differentiation of HL-60 cells.
In vivo, the administration of DADS to SCID mice bearing HL-60 cells caused significant growth suppression of tumors at dose (42 mg/kg) with little
detectable toxicity. Some studies have indicated that DADS inhibits the proliferation of human colon tumor
cells[22,23], DS19 mouse erythroleukemia cells and K562 human leukemia
cells[24] by increasing acetylated histone
in vitro. In the present study, we found that DADS caused the increase of acetylated histone in HL-60 cells cultured with the agent within 24 h. The
increase of acetylated histone was observed in the HL-60 cell xenografts after the administration of 42 mg/kg DADS by ip
injection 3 times a week in SCID mice. The effects were similar to that of SB, which is a potent histone deacetylase inhibitors
(HDI)[31]. Consequently, we presume that DADS may serve as a potential HDI and inhibits HL-60 cell growth. HDI have been
reported to induce G1 or G2 phase arrest and regulate the transcription of a number of cell cycle regulator genes, including p21,
c-myc, cyclin and cdk[32_34]. HDI are currently receiving considerable attention as antitumor agents because of their ability to
induce cell cycle arrest and/or cell death in a wide range of transformed cells
in vitro and in
vivo[35,36]. Our previous work shows that DADS induced cell cycle
arrest at the G0/G1 phase on HL-60 cells
in vitro[29]. Here we found that DADS also induced cell cycle arrest at the
G0/G1 phase on HL-60 cells in SCID mice
in vivo. The cycle-dependent kinase inhibitor
p21WAF1 has been identified as a target induced by
HDI in transformed cells[37]. The expression of
p21WAF1 in transformed cells after treatment with HDI was preceded by
localized hyperacetylation of histones in the chromatin region containing the
p21WAF1 gene[38,39]. These findings suggest that
these agents act directly to induce hyperacetylation and thereby alter the chromatin structure in the region of the
p21WAF1 gene. Our results show that DADS could induce hyperacetylation of histones and increase the expression of
p21WAF1 in HL-60 cells. We then presume that DADS has an antitumor effect on HL-60 cells
in vitro and in vivo because it could possibly
cause hyper-acetylation of histones in the promoter region of the
p21WAF1 gene. However, this requires further confirmation
by chromatin immunoprecipitation assay.
In conclusion, DADS significantly inhibits the growth of human acute myeloid leukemia HL-60 cells
in vitro and in vivo. The anticancer mechanism may involve the increase of histone acetylation, upregulation of
p21WAF1 and G0/G1 phase arrest.
DADS may be a potent antitumor agent for the management of human acute myeloid leukemia.
Acknowledgment
We would like to thank Lea MA (Department of Biochemistry and Molecular Biology, UMDNJ _ New Jersey Medical
School, Newark, NJ, USA) for criticism and encouragement.
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