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  Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 51  |  Issue : 4  |  Page : 609-613
 

Effect of aged garlic extract on immune responses to experimental fibrosarcoma tumor in BALB/c mice


1 Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, Iran
2 Infectious Diseases and Tropical Medicine Research Center, Babol University of Medical Sciences, Babol, Iran

Date of Web Publication1-Feb-2016

Correspondence Address:
M Abouhosseini Tabari
Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-509X.175359

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 » Abstract 

Background: Aged garlic extract (AGE) has many biological activities including radical scavenging, antioxidative and immunomodulative effects. Aim: In this research work, the antitumor and immunomodulatory effects of AGE against fibrosarcoma implanted tumor were studied. Materials And Methods: WEHI-164 fibrosarcoma cells were implanted subcutaneously on day 0 into the right flank of 40 BALB/c mice at age of 8 weeks. Mice were randomly categorized in two separate groups: First received AGE (100 mg/kg, IP), second group as the control group received phosphate buffered saline. Treatments were carried out 3 times/week. Tumor growth was measured and morbidity was recorded. Subpopulations of CD4+/CD8+ T cells were determined using flow cytometry. WEHI-164 cell specific cytotoxicity of splenocytes and in vitro production of interferon gamma (IFN-γ) and interleukin-4 cytokines were measured. Results: The mice received AGE had significantly longer survival time compared with the control mice. The inhibitory effect on tumor growth was seen in AGE treated mice. The CD4+/CD8+ ratio and in vitro IFN-γ production of splenocytes were significantly increased in AGE group. WEHI-164 specific cytotoxicity of splenocytes from AGE mice was also significantly increased at 25:1 E: T ratio. Conclusion: Administration of AGE resulted in improved immune responses against experimentally implanted fibrosarcoma tumors in BALB/c mice. AGE showed significant effects on inhibition of tumor growth and longevity of survival times.


Keywords: Aged garlic extract, BALB/c mice, fibrosarcoma, tumor therapy


How to cite this article:
Tabari M A, Ebrahimpour S. Effect of aged garlic extract on immune responses to experimental fibrosarcoma tumor in BALB/c mice. Indian J Cancer 2014;51:609-13

How to cite this URL:
Tabari M A, Ebrahimpour S. Effect of aged garlic extract on immune responses to experimental fibrosarcoma tumor in BALB/c mice. Indian J Cancer [serial online] 2014 [cited 2019 Aug 24];51:609-13. Available from: http://www.indianjcancer.com/text.asp?2014/51/4/609/175359



 » Introduction Top


Cancer is accounted for 7.6 million deaths globally in 2008 and it is estimated to continue to rise to over 11 million in 2030. Different therapeutic formulations are available for different cancers based on the types and stages of cancer and the tissue involved. Although common therapies, like chemotherapy, are accompanied with numerous side-effects,[1],[2] supplement therapy of cancers using dietetic ingredients, like alpha lipoic acid [3],[4] or medicinal herbs, like aged garlic extract (AGE),[5] may results in enhanced efficacy of the generic therapies and improved life quality for cancer patients. In addition, aged garlic can reduce the negative side-effects of chemotherapy agents.

Garlic, Allium sativum, is a species of the onion family, Alliaceae. It has been extensively used throughout history for its prophylactic and therapeutic effects. Its immunomodulatory and antitumor effects have also been demonstrated by in vitro and in vivo experiments. Garlic extract contains more than 200 chemicals with diverse properties. Different chemicals were detected in garlic using different extraction procedures. Garlic also shifts polarization of CD4+ T cells toward T helper cells 1 (Th1),[6],[7] increases frequency and function of natural killer (NK) cells,[6],[8],[9],[10],[11],[12],[13] enhances frequency and proliferation potential of lymphocytes,[14],[15] improves CD4+/CD8+ T cell ratio,[16] has anti proliferative and anti-angiogenesis effects on tumor cells,[12],[17],[18] modifies monocyte/macrophage function upon endotoxin stimulation through involving in the tool like receptor signal transduction events,[19] has anti apoptotic properties [2],[20],[21] and demonstrates high antioxidant activities.[22]

Several purified chemicals from garlic or its crude extracts have been demonstrated to have obvious antitumor activities.[23],[24],[25],[26] Direct pre-exposure of tumor cells with garlic extracts resulted in complete growth inhibition of implanted tumor cells.[27] It is believed that malignancies are accompanied with increased production and release of potentially harmful free reactive oxygen species (ROS) and AGE has antioxidant potential to scavenging the ROS. Furthermore, AGE acts as an enhancer of cellular antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase activities and also increases glutathione levels in the cells.[27],[28],[29] Antioxidant constituents of garlic result in enhanced antioxidant capacity of the body, improved immunity and effective scavenging of free radicals.[31] Increased nitric oxide synthase activities of endothelial cells and neutrophiles [35],[31] and also anti-teratogenic properties of garlic have been demonstrated.[32] Golovchenko et al. studied the mechanisms contributing to antiangiogenic properties of garlic. Methylallyl thiosulfinate, a constituent of garlic extract, was shown to have an inhibitory effect on platelet-derived growth factor-induced vascular smooth muscle cell migration, via inhibition of geranylgeranyl transferase I.[18]

The present study investigated the effects of administration of AGE on experimental fibrosarcoma cell tumors in BALB/c mice. Survival times, tumor volumes, CD4+/CD8+ T cell ratios in spleen, tumor specific cytotoxicity of splenocytes and in vitro production of interferon gamma (IFN-γ) and interleukin 4 (IL-4) cytokines were assayed and compared.


 » Materials And Methods Top


Female inbred BALB/c mice and WEHI-164 BALB/c mouse fibrosarcoma cell line were purchased from Pasteur Institute, Tehran, Iran. Other reagents used for in vitro measurements were as follows: Naltrexone hydrochloride (Sigma), fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (Serotech), phycoerythrin (PE)-conjugated anti-CD8 (Serotech), lactate dehydrogenase (LDH) release kit (Roche-applied), IL-4and IFN-γ cytokines kits (Quantikine).

Garlic bulbs were peeled and minced in an aqueous-alcoholic solution and kept under anaerobic conditions for 8 months. Aged garlic was crushed using a mortar and pestle and homogenized in distilled water. The homogenized preparation was filtered through Whatman paper No. 1 and the filtrate was centrifuged at 4500 G for 30 min. The cleared supernatant was collected and used. The AGE, (containing 0.4 g of garlic materials/ml) was diluted in distilled water and 100 mg/kg of the preparation was administered intraperitoneally to each mouse.

WEHI-164 cell line was used for generation of tumor in mice and as a target cell for cytotoxic T lymphocytes assay. The cells were cultured using RPMI 1640 (GIBCO) media, supplemented with 10% heat inactivated fetal bovine serum (FBS) (FBS, GIBCO), 100 μg/ml streptomycin and 100 U/ml penicillin (GIBCO) and incubated in 37°C in a humidified, 5% CO2 atmosphere. The cells in logarithmic growth phase were used to establish tumor model by subcutaneously implanting of 1 × 10^6 cells/200 μl into the right flank of mouse. Tumor dimension was measured on 7 days intervals using vernier caliper. Tumor volume (mm 3) was calculated by the formula: Length × width2 × π/6.

A total number of 40 BALB/c mice at 6-8 week of age went under generation of experimental tumor by injecting of WEHI-164 cells obtained from logarithmic phase of the cell line culture on day 0. Tumorized mice were randomly divided into two groups, 1st received AGE (100 mg/kg, IP), 2nd group (control or phosphate buffered saline [PBS] group) received only PBS. All injections were performed in alternate days intervals (3 times/week) until the time for mouse killing (for in vitro study) or dying (for survival analysis). Half of the mice in each treated group were considered for survival study and tumor volume measurements and the others were euthanized on day 28 (contemporary with the first mortality in PBS group) for in vitro studies of immunologic parameters. All procedures on animals were in accordance with Pasteur Institute of Iran, laboratory animal care and use law.

For survival analysis mortalities of tumor bearing mice were recorded with respect of time. Tumor volumes were also recorded within weekly intervals.

For in vitro studies mice were euthanized on day 28 and the splenocytes were isolated as a single-cell suspension. Erythrocytes were then lysed at room temperature using ammonium-chloride-potassium lysis buffer (NH4 Cl, KHCO3, Na2 EDTA). The isolated splenocytes were used for in vitro measurements after three steps washing with PBS. The CD4+ and CD8+ T cells percentages, specific cytotoxicity of splenocytes against WEHI-164 cells and in vitro production of both IL-4 and IFN-γ cytokines were determined.

The percentages of T CD4+ and T CD8+ cells of freshly isolated splenocytes were determined. Viability of isolated cells was determined by trypan blue exclusion method. The cells were resuspended in RPMI 1640 (GIBCO) supplemented with 10% fetal calf serum (GIBCO). The freshly prepared cells were analyzed by flow cytometry after immunostaining with two fluorochrome labeled antibodies (FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 antibodies, both from Serotech). Each sample was immunostained with the antibodies for 45 min at 4°C. The cells were washed in washing buffer and fixed with 2% paraformaldehyde. The percentage of CD4+ and CD8+ T cells was determined by flow cytometric analysis of immunostained cells using an EPICS flow cytometer (Coulter).

The cytolytic activity of splenocytes was measured by LDH release assay. Single cell suspension of splenocytes was prepared and used as effector cells. WEHI-164 cells (1 × 10^4 cells/100 μl) as target cells were incubated with 100 μl effector cells suspension at effector/target ratios of 1:10, 1:25 or 1:50. LDH release assaying kit was used (according to the instruction of manufacturer, Roche-Applied) to measure LDH enzymes released from lysed cells. For low and high control wells (spontaneous release and maximum release, respectively) 100 μl of assay medium or %2 Triton X100 in assay medium was added. All experiments were performed in triplicates and percentage of specific cytotoxicity was determined by the following formula:



Cytokine releases after in vitro stimulation of splenocytes with tumor cell lysate were measured using standard enzyme-linked immunosorbent assay (ELISA) kits. Splenocytes at the concentration of 1 × 10^6 cells/well were cultured in the 24 well plates in the presence of 50 μg/ml WEHI-164 cell lysate (prepared by sonication of the cells), 8 μg/ml PHA or complete media, in the total volume of 1 ml. RPMI 1640 supplemented with 10% heat inactivated FBS, 100 μg/ml streptomycin, 100 U/ml penicillin was used. The plates were incubated for 48 h at 37°C in a humidified 5% CO2 atmosphere. Supernatants were collected after centrifugation, IFN-γ and IL-4 concentrations were determined using commercial sandwich ELISA assay kits (Quantikine) according to the manufacturer's instructions.

Results from triplicate measurements were averaged. Statistical analysis was performed using the Student's t-test. Kaplan-meier analysis was applied to compare survival time between groups and log-rank test was used to pairwise comparisons. A value of P < 0.05 was considered to be statistically significant. All statistical analyses were conducted with SPSS 16 software.


 » Results Top


Only tumor bearing mice were entered the study. Environmental conditions were the same for all mice. The day of tumor implantation was considered as zero for survival time records and treatment programs. One day after tumor implantation animals were randomly assigned to treatment groups (two groups each 2 × 10 mice) and treatments were initiated. Survival analysis was conducted in parallel with in vitro study. Survival records were terminated when the last living mouse was lost. The in vitro study was begun at day 28 when the first mouse died of tumor.

Survival analysis was done for tumor bearing mice had been treated using two different treatment protocols. Dying of each mouse as a terminal outcome was recorded with respect of time. Kaplan-Meier analysis and simultaneous log-rank test were applied for total and pair wise comparisons. The increase in life span (ILS) of the treated groups was expressed as a percentage (%ILS). Median survival times (MST) was determined and the %ILS was calculated as: %ILS = {(MST [days] of treated mice/MST [days] of control mice) − 1} ×100. Garlic treated group had increased ILS (%60.6). Mean ± SE of survival times (day) in AGE treated group was 52.4 ± 2.1 and in the control group was 35 ± 1.8.

Tumor volume was calculated as mean ± SE for all mice and expressed in mm 3. All mice in the control group had palpable tumors on day 14 post implantation while the percent of tumor bearing mice in AGE was %2. Garlic exerted inhibitory effects on tumor growth [Figure 1].
Figure 1: Tumor volume (mm3) with respect of time (day) stratified by treatment. *Significant difference in comparison to phosphate buffered saline group (P < 0.01)

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AGE treated mice had increase in the spleen lymphocyte subpopulations ratio compared with the control group, but the differences were not statistically significant (P = 0.62). Amount of CD4+/CD8+ ratio in AGE treated mice was 4.85 ± 0.51 and in the control group it was 3.6 ± 0.36 [Figure 2].
Figure 2: Percent cytotoxicity of splenocytes against WEHI-164 cells at 3 E:T ratio

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The splenocytes from the mice were analyzed for in vitro production of IFN-γ and IL-4 cytokines due to stimulation by exposure to WEHI-164 cell lysate. The splenocytes of AGE receiving group showed significant (P < 0.05) increase in the level of IFN-γ comparing to control group [Figure 3]. There were no noticeable differences between groups considering the in vitro IL-4 production level [Figure 4].
Figure 3: In vitro interferon gamma production of splenocytes after exposure to WEHI-164 cell lysate. *Significant difference in comparison to phosphate buffered saline group (P < 0.05)

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Figure 4: In vitro interleukin-4 production of splenocytes after exposure to WEHI-164 cell lysate

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 » Discussion Top


Cancer is the second leading cause of death in the United States. It's estimated to be the leading cause of death within the next decade. Selective therapies for cancer patients depend upon the type of cancer, its stage and its proximity to other organs. Garlic has well-known anti-cancer properties.

Chronic inflammation and continuous oxidative stress may result in genetically modified cells that may finally transform to cancerous cells. Various cancers also result in depletion of body's antioxidant resources and lead to unresolved oxidative stress that can further damage cells and tissues and help the existing tumor readily survive. In diseases other than cancers that accompanying with chronic oxidative stress the incidence of cancer are also noticeably high. In other words, proper facing on production and release of free radicals and their harmful products, like nitrosamines, is the central step in fighting against cancer.[25],[33],[34],[35]

Garlic resolves the oxidative stress firstly by its antioxidant ingredients and secondly by enhancing pre-existing antioxidant systems of the body.

Hodge et al. has reported that application of fresh garlic extract by the in vitro culture methods resulted in inhibition of Th1 and inflammatory cytokines production by peripheral blood mononuclear cells while the production of Th2 cytokine, IL-10, was up regulated. Augmenting of Th2 cytokines is probably because of the fresh garlic application in the study. Fresh garlic preparations have usually few antioxidants and make of different ingredients compared to AGE.[36] Kasuga et al. also mentioned in his report that different types of garlic preparations have different pharmacologic properties and among the four garlic preparations studied, AGE could be the most useful garlic preparation.[11]

Upon the occurrence of any mutated and uncontroled cells in the body, this is the commitment of cells contributing in the immune system responses to recognize and efficiently destroy it. Garlic modulates the immune responses by different pathways. Garlic increases antioxidant capacity of the cells and prepare improved circumstances to proper deciding and acting against threats. In this way, garlic exerts its modulatory effects on innate and adaptive immunity.[37] Intraperitoneal administration of AGE has led to increased macrophage counts and enhanced killing activities of the cells.[38] Dietary administration of garlic has led to elevation of white blood cell counts [39] and enhanced NK activity in peripheral bloods of animal models. Garlic also modifies productions of cytokines by immune cells.[19],[40],[41]

In the present study, AGE was used to inhibit fibrosarcoma tumor growth in BALB/c mice. AGE receiving group showed significantly retardation in tumor appearance. All PBS treated control mice showed visible tumors (%100) 14 days post-implantation. Administration of 100 mg/kg of AGE efficiently inhibited tumor growth and significantly increased ILS of the mice in comparison to control mice.

Von Euler et al. have reported for the first time in 1947, growth-inhibiting activity of pure allicin an active component of garlic, on various tumors, with inconsistent complete inhibition in rats of Jensen sarcoma and of a benzpyrene-induced sarcoma. Fujiwara and Natata also has tried to induce tumor immunity using tumor cells treated with extract of garlic.[42] Slight delay in tumor appearance and animal death through administration of garlic extract has also been reported by Aboul-Enein in 1986.[43] It has also been shown by Hu, et al. using an in vitro assessment that direct exposure of tumor cells with AGE resulted in suppression of tumor cell growth and inhibition of the cell migration. They concluded that garlic as a natural plant may play a role in fighting against cancer without significant side-effects.[27] Present study with AGE (IP) administered 1 day after tumor implantation also showed significant retardation in tumor appearance and ILS (%60.6 compared with the control group).

For preliminary elucidation of the mechanisms involving in mouse resistance against the tumor, we assayed several immune parameters using splenocytes. There was no significant difference between AGE and control groups (P > 0.05). Low ratio was seen in AGE treated mice probably was the result of increases in both CD4+ and CD8+ T cells, thus the ratio undergo minimum changes. Present study showed increased cytotoxic activity of splenocytes from AGE treated mice but the differences were not statistically significant.

IFN-γ secretion by CD4+ Th1 cells, CD8 cells, gamma/delta T cells and activated NK cells plays an important role in activating lymphocytes to enhance anti-microbial and anti-tumor effects. We found a significant increase in IFN-γ production of splenocytes from AGE treated mice. It seems garlic administration has a profound effect on IFN-γ production of splenocytes. Our findings showed that the administration of AGE induced effective immune responses against fibrosarcoma tumor in BALB/c mice and led to significant inhibition of tumor growth and enhanced survival times of mice.

 
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