Antiproliferative property of aqueous extract of garlic on human larynx tumour and non-tumour mouse fibroblast cell lines.Introduction: Laryngeal cancer is one of the most common head and neck malignancies and has been reported to account for approximately 1% of all new cancer diagnoses. Experimentally garlic and its associated sulfur components are reported to reduce tumour incidence in breast, colon, skin, uterine, esophagus and lung. In this study we investigated the in vitro effects of aqueous extract of garlic on Hep2 human larynx tumour cell lines as case and L929 cell lines as control.
Materials and methods: In a morphologic examination, Hep2 and L929 cell lines were cultivated in a 1:1 mixture of DMEM DMEM Dulbecco's Modified Eagle's Medium (for cell culture growth)
DMEM Design Manufacture and Engineering Management Department , supplemented with 10% FCS FCS - Frame Check Sequence and 1% penicillin-streptomycin solution. Each cultured cell line was seeded into seven 50 [cm.sup.2]-flasks and after 24h both groups of cells were incubated with different concentrations [0 (control), 0.5, 1, 4, 8, 10 and 12 mg/mL] of the extract. Cells were observed under light inverted microscope for morphological changes at 24, 48 and 72h. In a MTT assay, Hep2 and L929 cell lines were cultivated in three 96-well plates (3 x103 cells/well) and the extract was added to growth medium at concentrations of 0 (control), 0.5, 1,4, 8, 10 and 12mg/mL.
Results: After 24, 48 and 72h one plate was selected and cell proliferation was determined using MTT assay. After 24h, morphological changes in both cells showed growth inhibitory effects at concentrations of 10 and 12 mg/mL of the extract. These changes became more prominent after 48 and 72h. MTT assay results indicated a significant decrease in percentage of surviving cells between Hep2 and L929 cells incubated in concentrations of 8, 10 and 12 mg/mL and the cells incubated in the absence of the extract.
Conclusion: The results of this study demonstrate that this type of garlic extract exerts antiproliferative effects on both Hep2 and L929 cell lines.
Keywords: Antiproliferative, aqueous extract, garlic, Hep2, L929, MTT MTT 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide
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Laryngeal cancer is one of the most common head and neck malignancies and has been reported to account for approximately 1% of all new cancer diagnoses (Makitie 1999). Almost all laryngeal cancers (90-95%) are squamous cell carcinoma squamous cell carcinoma
A carcinoma that arises from squamous epithelium and is the most common form of skin cancer. Also called cancroid, epidermoid carcinoma. (SCC SCC - strongly connected component ) (Almadori 1996).
Garlic (Allium sativum) has been known as a potent medical plant for centuries (Ghazanfari 2002). Experimentally garlic and its associated sulfur components are reported to reduce tumour incidence in breast, colon, skin, uterine, esophagus and lung (Milner 2001). It seems that the anticancer effect of garlic components is a consequence of their direct cytostatic cytostatic /cy·to·stat·ic/ (sit?ah-stat´ik)
1. suppressing the growth and multiplication of cells.
2. an agent that so acts.
1. or cytotoxic properties (Colic 2002). When garlic is cut, chopped or crushed, the clove membrane is disrupted and Sallylcysteinesulfoxide (alliine) is transformed enzymatically into allicin allicin /al·li·cin/ (al´i-sin) an oily substance, extracted from garlic, which has antibacterial activity.
allicin by alliinase (Bianchini 2001). Allicine has been reported to inhibit the proliferation of several human malignant cells.
Ajoene is a garlic derived compound produced most efficiently from pure allicin and has the advantage of a greater chemical stability. Recently ajoene was shown to inhibit proliferation and induce apoptosis of human leukemia CD34-negative cells (Ahmed 2001). No reports are available on the effects of aqueous extract of garlic on SCC. Our present studies therefore were designed to examine the effects of this extract on the in vitro proliferation of Hep2 cells (SCC type2), originated from laryngeal carcinoma. The L929 cell line was studied as a control group.
Materials and methods
Preparation of extract: Fresh whole plants of garlic were collected from a farm in Hamedan, Iran. The plant cloves were crushed by knife and aqueous extract was prepared using 50 gm crushed cloves and 400 mL distilled water in Soxhelet set for 17h. The volume of this extract was reduced to 100 mL by a buchi evaporator under reduced pressure. Then the extract was sterilised using 0.2-gin filter. Sterile extract 10 mL was transferred to tarred dishes and dried on a water bath. The solid sterile extract was weighed at a concentration of 129 mg/mL.
Morphologic examinations: Hep2 and L929 cell lines were provided from national cell bank of Iran (NCBI NCBI National Center for Biotechnology Information (NIH)
NCBI National Coalition Building Institute
NCBI National Council for the Blind of Ireland (Dublin, Ireland) ). Cells were cultivated in a 1:1 mixture of Dulbeccos Modified Eagles Medium (DMEM), supplemented with 10% fetal calf serum and 1% penicillin-streptomycin solution (Durmaz 1999). The cultured cells were humidified at 37[degrees]C and 5% Co2 atmosphere and fed 2-3 times a week until they approached confluence (Chiba 1998; Durmaz 1999). Cell aggregates were treated with 0.25% trypsin-EDTA solution and incubated 10-15 rain at 37[degrees]C when cells were mechanically dispersed using a 10 mL pipette. Trypsin trypsin, enzyme that acts to degrade protein; it is often referred to as a proteolytic enzyme, or proteinase. Trypsin is one of the three principal digestive proteinases, the other two being pepsin and chymotrypsin. activity was inhibited by adding growth medium and cells were centrifuged at 1000 rpm for 5 rain. Supernatant was removed and pellets of each cell line were seeded into seven 50 [cm.sup.2] flasks containing 5x [10.sup.5] cells. Under these conditions cell viability was found to be higher than 95% by trypan blue exclusion testing. After 24h (Chiba 1998, Whelan 2003) both group of cells were incubated by different concentrations [0 (control), 0.5, 1, 4, 8, 10 and 12 mg/mL] of the extract. Cells were observed under light inverted microscope for shape, granulation granulation /gran·u·la·tion/ (-shun)
1. the division of a hard substance into small particles.
2. the formation in wounds of small, rounded masses of tissue during healing; also the mass so formed. and suspension (anchorage independency) at 24, 48 and 72h. Morphologic examination was repeated 3 times.
MTT assay: The effect of aqueous extract of garlic on proliferation Hep2 and L929 cell lines was determined using 3- (4, 5- dimethyl thiazol -2-yl) 2, 5-diphenyl tetrazolium bromide (MTT) colorimetric col·or·im·e·ter
1. Any of various instruments used to determine or specify colors, as by comparison with spectroscopic or visual standards.
2. assay, originally described by Mosman (1983) and modified by Alley et al (1988) (Durmaz 1999). For this examination three 96-well plates were prepared in this way: 21 (3 x 7) wells of each plate were selected and Hep2 cells were seeded (3 x [10.sup.3] cells/well). In the same way L929 cells were cultivated in another 21 (3 x 7) wells of each plate. The cultures were humidified at 37[degrees]C and 5% Co2 atmosphere for 24h. At the end of this procedure culture medium of each plate was changed by fresh culture medium. The extract was added to growth medium at concentrations of 0 (control), 0.5, 1, 4, 8, 10 and 12mg/mL such that each 3 wells were incubated by one of these concentrations. After 24h one plate was selected randomly and cell proliferation was determined using MTT assay. For each 200 [micro]l growth medium, 20 [micro]l MTT solution (Sigma) was added and incubated for 4h (Durmaz 1999). After all growth mediums had been removed, crystal particles of MTT were dissolved by adding 200 pl dimethylsulfoxide di·meth·yl·sulf·ox·ide
DMSO. (DMSO DMSO dimethyl sulfoxide.
Dimethyl sulfoxide; a colorless hygroscopic liquid obtained from lignin, used as a penetrant to convey medications into the tissues.
n. ) and 25 gl glycin while plates were shaken for 2-3 min. The absorbance absorbance /ab·sor·bance/ (-sor´bans)
1. in analytical chemistry, a measure of the light that a solution does not transmit compared to a pure solution. Symbol .
2. of formazan dye was read at 570 nm using ELISA ELISA (e-li´sah) Enzyme-Linked Immuno-Sorbent Assay; any enzyme immunoassay using an enzyme-labeled immunoreactant and an immunosorbent.
n. plate reader (Chiba 1998, Abe 2000). All the procedures were repeated in the same manner for the second and third plates after 48 and 72h.
Statistical analysis: Data was analysed with one way analysis of variance (ANOVA anova
see analysis of variance.
ANOVA Analysis of variance, see there ) followed by Tukeys multiple range tests for honestly significant differences. The values were considered statistically significant if p<0.05.
Both Hep2 and L929 cells incubated with low concentrations (0.5, 1 and 4mg/mL) of aqueous extract of the garlic in the sense of morphology were very similar to their controls (cells incubated in the absence of the extract) after 24h. Morphological effects of the extract on Hep2 cells started at concentration of 8 mg/mL but these effects on L929 started at concentration of 10 mg/mL. Hep2 cells were completely affected at concentrations of 10 and 12 mg/mL and L929 cells were completely affected at concentration of 12 mg/mL; cells were round and suspended with increased granulation of cytoplasm when compared to the control. These morphological changes became more prominent after 48 and 72h incubation (figure 1).
Optical density (OD) of Hep2 and L929 cells incubated with different concentrations of garlic extract was compared to OD of the control cells and the percentage of surviving cells was determined as follows: The percentage of surviving Hep2 cells at 8 mg/mL and 10mg/mL of the extract was significantly decreased when compared with control cells (both p<0.05) after 24h. There was a correlation between increase of the extract concentration and decrease of surviving Hep2 cells (r = -0.823). There was also a correlation between increase of the extract concentration and decrease of surviving L929 cells (r = -0.823) after 24h. These changes became more prominent after 48 and 72h incubation. A significant decrease in percent of surviving cells of Hep2 (P<0.001) and L929 (P<0.005) was present at 8, 10 and 12mg/mL concentration of the extract by 72h (figure 2).
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
To our knowledge no previous literature has reported on the effect of aqueous extract of garlic on Hep2 and L929 cells. This investigation clearly demonstrates the inhibitory effect of aqueous extract of garlic on growth of human larynx cancer cell lines (Hep2). According to both morphologic and MTT assay in this study the inhibitory effects started at concentration of 8 mg/mL and higher from 24h and were more prominent at 48h and 72h. The strongest inhibitory effects were present at 72h. The data also demonstrates the inhibitory effect of aqueous extract of garlic on growth of L929 cells that start at a concentration of 10 mg/mL. The maximum inhibitory effects of the extract on growth of L929 cell lines were present at a concentration of 12 mg/mL. The inhibitory effects on growth of cancer cells in human and animals for crude garlic both aqueous and alcoholic extracts and different isolated compounds of garlic especially organosulfure have been reported by different investigators (Li 2002, Sundaram 1993).
The inhibitory effects of aqueous extract of garlic may be due to inhibition of gene mutation, suppressing the formation of DNA-adducts, change of enzyme activity and induction of apoptosis as was reported by Bianchini et al (2001). Cancer development is known to be associated with increased cell proliferation and decreased apoptosis. On the other hand induction of apoptosis may insert a suppressive sup·pres·sive
Tending or serving to suppress.
Adj. 1. suppressive - tending to suppress; "the government used suppressive measures to control the protest" effect on cancer development. Induction of apoptosis by garlic on a buccal pouch carcinoma in hamster (Balasenthil 2002) and in human tumour cells (Karasaka 2001) has been reported. The cytotoxic effect of garlic in the present study may also be due to this mechanism. Administration of garlic has been shown to prevent carcinogen induced genetic damages (Sengupta 2002). As apoptosis can also occur in non tumour cells, this mechanism may argue against the antiproliferative effect that was demonstrated on L929 cell line in the present study. More investigations are required to ascertain the mechanisms of inhibitory actions of aqueous extract of garlic on cell growth.
We have demonstrated, using cell culture model, an antiproliferative affect of aqueous extract of garlic on human larynx turnout (Hep2) and L929 cell lines. As antitumour effects of garlic components have been shown in many studies and because of the antiproliferative effect of this extract on non-turnout L929 cells, these results indicate that use of another type of garlic extract may be better than Soxhelet aqueous extract for treatment and prevention of cancer.
This study was supported by Booali research institute from Mashhad University of Medical Sciences Mashhad University of Medical Sciences (MUMS) is a medical school in Iran.
Located in Razavi Khorasan province in the city of Mashhad, it was established in 1949 along with Ferdowsi University of Mashad, and separated in 1986 from its parent institution by national , Iran.
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Department of Physiology
Mashhad University of Medical Sciences Islamic
Republic of Iran
Phone: +98 0511 8440350
Fax: +98 0511 8413579
Jalil Tavakkol Afshari
Immunology and cell culture laboratory, Immunology research center, Booali research institute
Mashhad University of Medical Sciences Islamic
Republic of Iran
Phone: +98 0511 7112611
Fax: +98 0511 7112616
Email: Jtavakkol @yahoo.com
Department of Physiology, Mashhad University of Medical Sciences, Islamic Republic of Iran.
Phone: +98 09153148741 and +98 0511 8413579
Fax: +98 0511 8413579
Mohammad Taghi Shakeri
Department of Social Medicine
Mashhad University of Medical Sciences Islamic Republic of Iran.
Phone: +98 0511 8400494
Fax: +98 0511 8684137