PROLIFERATION INHIBITION OF LEUKEMIC CELL LINES BY DIALLYL
DISULFIDE (DADS)
1,
2
#
*e-mail: lukpla_24@hotmail.com, #e-mail: dalina.itc@mahidol.ac.th
Abstract
Medicinal plants have been used as alternative medicine for cancer treatment.
However, there is less information about leukemia. Diallyl disulfide (DADS), which is a organosulfur compound from crushed garlic, has effect on anti-inflammation and anti-cancer.
In this study, the effect of DADS on cell viability, cell proliferation inhibition and induction apoptosis were studied in K562 and NB4 leukemic cell lines. Leukemic cell lines were treated with DADS at concentration 100, 200, 300 and 500 µg/ml for 24h and 48h. Cell viability, cell inhibition and percent cell apoptosis were analyzed by trypan bule staining,
MTT assay and flow cytometry, respectively. In addition, mTOR inhibitor was used to investigate the involvement of mTOR pathway in leukemic cell proliferation. The results showed that DADS decreased cell viability with dose and time dependent manner and DADS increased percent cell inhibition and cell apoptosis in both cell lines. The highest percent cell apoptosis was found in 300 µg/ml treated cells at 48h. Moreover, mTOR pathway is involved in proliferation of leukemic cell line. From results of this study suggest that DADS could be developed as candidate drug for leukemia treatment in the future.
Keywords: Leukemia, Diallyl disulfide, Proliferation inhibition
Introduction
Leukemia is one of hematological malignancy. The leukemia is uncontrolled proliferative of hematopoietic cells especially leukocyte precursor. The leukemia classification base on hematopoietic lineages; myeloid and lymphoid leukemia, which follow criteria of World Health Organization (WHO) and French American British (FAB) (1). An estimated a total of 144,559 leukemia cases were identified 66,067 (46%) has acute and
71,860 (50%) has chronic leukemia in North American (2). In Shanghai, China the incidence of ALL was 0.81 in 100,000 of sample from 2002-2006 (3).
The comparing of cancer mortality below the age of 40 years old in Asia by used WHO database, in female the leukemia was ranked first in the four countries; Japan, Korea, Philippine and Uzbakiztan.
Treatment for leukemia has various approaches such as chemotherapy, radiotherapy and stem cell transplantation. However, they have many side effects, include nausea and vomiting from chemotherapy, mouth sores followed by nausea and vomiting were 42% of the patients treatment by bone transplantation (4). Then alternative medicine focusing in herbs and herbs compounds were developed. Many studies have been report the effect of herbs extract on many types of cancer including leukemia. For example, the herbal compound isolated from plant Withania somnifera has effectively induces apoptosis in myeloid (K562) and lymphoid
(MOLT-4) cell lines (5). Allicin is compound from garlic could induce apoptosis in cancer cells (6). Many evidences show garlic consumption has an antitumorigenic potential in several types of cancer such as stomach cancer, colon cancer, breast cancer and lung cancer
(7). Diallyl disulfide (DADS) is one of sulfur compounds in garlic oil (Figure 1) (8). DADS has an effect on induction apoptosis in human leukemia HL-60 cell line via activation of caspase-3 (7) and induce cytotoxicity to WEHI-3 leukemia mice also induce the differentiation of immature cells (9). The previous reports suggest that DADS involve in

many pathways such as apoptosis pathway, MAPK kinase pathway including PI3K pathway.
Mammalian target of rapamycin (mTOR) is a protein kinase and belong to PI3K family, play role in cell survival and also in cell cycle. It can stimulated by insulin growth factor and energy level. Many scientists interesting on mTOR inhibitor for treatment hematological malignancies (10, 11).
However, There is less information about mechanism of DADS on induction inhibition proliferation of leukemic cells .
Materials and Methods
Reagents
RPMI-1640 medium, penicillin-streptomycin and fetal bovine serum purchased from Invitrogen, USA. Diallyl disulfide (DADS) purchased from Sigma Aldrich.
Annexin V-FITC apoptosis detection kit purchased from BD biosciences. PP242 mTOR inhibitor purchased from Millpore and dissolved in distilled water.
Cell culture
Human chronic myelogenus leukemia cell line (K562) and human acute promyelocytic leukemia cell line (NB4) were purchased from cell line service (CLS).
Leukemic cell were cultured with RPMI-1640 medium supplement with 10% fetal bovine serum (FBS) and 2% penicillin-streptomycin. Cells were grown at 37
๐ c with 5% Co
2
.The medium was changed every 3 days.
Cell viability assay
Leukemic cell were incubated with diallyl disulfide (DADS) at concentration
100,300 and 500 µg/ml with and without mTOR inhibitor at various concentration (0,1,5,10
µM) for 24 h and 48 h. Then cell were stained with 0.4 % trypan blue in distilled water at
24h and 48 h. Count viable cells under light microscope. Calculated a total cell count and present of cell viability.
MTT assay
Seed cell 10
4
cell/well in 96 well plate and incubate with various concentration of DADS (0,100,200,300 and 500 µg/ml) for 24 and 48 h. Then 50 µl of MTT reagent was added into each well and incubated 4 h. After that, 100 µl of solubilize was added into each well and incubated the plate overnight.The absorbance was measured at wave length 570 nm by microplate spectrophotometer.
Cell apoptosis by flow cytometry
Leukemic cells were treated with DADS and washed with PBS twice and resuspend in 100 µl of 1x binding buffer. After cell were incubated with 5 µl of Annexin V-

FITC and propidium iodide (PI) for 15 minutes in dark. Then 350 µl of 1x binding buffer was added and measured apoptosis cell by FACSCanto II.
Results
Effect of DADS on cell viability
Leukemic cell lines, NB4 and K562 were incubated with various concentration of DADS for 24h and 48h. After that, cell were stained with 0.4 % trypan blue and calculated the percentage of cell viability. The results showed that DADS decreased cell viability in both of cell line with dose and time dependent manner. The lowest of cell viability was shown in cell treated with DADS at 300 µg/ml (Figure.1) . The IC
50
(half maximal inhibitory concentration 50%) of K562 cell line is 250 µg/ml and 120 µg/ml at 24h,
48h respectively. IC
50 of NB4 is 150 µg/ml at 24h and 100 µg/ml at 48h.
Effect of DADS on cell apoptosis
Figure 1. K562 (A) and NB4 (B) cell line treated with DADS at concentration 100,300,500 µg/ml for 24h and
48h. Cell viability was analyzed by trypan blue staining.

Effect of DADS on cell apoptosis
Leukemic cells were treated with DADS at concentration 100 and 300 µg/ for 48h.
The percentage of cell apoptosis determined by flow cytometry. The result showed that cell treated with DADS increase the percentage of cell apoptosis. The highest percentage of cell apoptosis was shown in 300 µg/ml of DADS at 48h for both cell line (Figure 2).
Figure 2. K562 (A) and NB4 (B) incubated with DADS at concentration 100,300 µg/ml for 24h and 48h. The percent cell apoptosis was measured by flow cytometry.
Effect of DADS on proliferation inhibition.
Leukemic cells were treated with DADS at concentration 0, 100, 200, 300 and 500
µg/ml for 24h and 48h. The proliferation inhibition was measured by MTT assay. The highest cell inhibition was shown in 500 µg/ml treated cells. Then DADS could inhibit cell proliferation with dose and time dependent manner ( Figure 3) .
Figure 3. K562 (A) and NB4 (B) incubated with DADS at concentration 100,200,300 and 500 µg/ml for 24h and 48h. The percent cell inhibition was analyzed by MTT assay.

Effect of DADS and mTOR inhibitor on proliferation inhibition.
To investigate of the mTOR pathway in leukemic cell proliferation, mTOR inhibitor was used for study its effect on cell viability. Leukemic cells were incubated with various concentration of DADS (0, 10,100 and 300
and mTOR inhibitor (0,1,5 and 10 µM) for 24h and 48h.The result suggest that mTOR inhibitor increased an effect of DADS to inhibit cell proliferation in both cell lines (Figure 4 ).

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