Farag et al
EFFECT OF THE MAGIC FRUIT EXTRACT ON TUMOR
INFILTRATING LYMPHOCYTES (TILS) PROPAGATED
EX VIVO
[1]
Farag, R. M. (1); Abdel-Hamed, S, (2); El-Housseini, M. E. (3)
and Selim, M. (3)
1) Faculty of Medicine, Ain Shams University. 2) Faculty of Science, Ain
Shams University. 3)Cancer Biology Dept., NCI, Cairo University.
ABSTRACT
Background: Tumor infiltrating lymphocytes (TILs) play a key role in the
immunogenic reaction against tumors but they are incapable of preventing
tumor growth. Pre-clinical data on ex-vivo studies revealed that TILs
activation have the ability to kill autologous cancer cells. The magic fruit
(Ziziphus jujube) or the Chinese date helps in many vital activities and
functions such as weight gain, improving liver function, stimulating the
immune system and others. Its role in the activation of TILs has not yet been
studied in Egypt and so the aim of the present work is to clarify the possible
effect of the extract on TILs in culture.
Patients and Methods: The present study was conducted on a total of 18
female patients with breast cancer taken from the outpatient clinics of the
NCI of Egypt. Tumor tissues were obtained during the period from January
2007 to August 2007. Tumor infiltrating lymphocytes (TILs) were
isolated and activated by IL-2. TILs were treated by the magic fruit extract
and counted before and after treatment. IFN- level was assayed in the culture
media before and after treatment, using a sandwich enzyme immunoassay.
Results: The mean value of the cell count after treatment by the magic fruit
extract was highly significant (p< 0.001) when compared to that before
treatment indicating an excellent effect of the extract on increasing the
number of cells after treatment. The mean value of IFN- after treatment was
also highly significantly more than that before treatment (p< 0.001). The
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correlations between both the cell count and IFN- with the
clinicopathological data were studied.
Conclusion: The treatment with the magic fruit extract exerted a significant
effect on the cell count and the level of IFN- in the culture media. These
results suggest casting more light on the use of the extract in activation of
TILs and returning them back to the patient as a type of cell therapy for breast
cancer.
INTRODUCTION
Tumor infiltrating lymphocytes (TILs) play a key role in the
immunogenic reaction against tumors (Whiteside and
Parmiani, 1994;
Kristen et al., 2005). Although TILs are present in the tumor
microenvironment and recognize the antigenic characteristics of malignant
cells, they are incapable of preventing tumor growth. However, pre-clinical
data on ex-vivo studies revealed that TILs activation has the ability to kill
autologous cancer cells (Topalian et al., 1989).
The magic fruit (Ziziphus jujube) or the Chinese date is a member of
Rhamnaceae family. It helps in many of vital activities and functions such as
weight gain, antiallergenic, improving stamina and strength, mildly sedating,
improving of liver function, stimulating the immune system, and helping to
efficiently get the energy from food and giving energy (Hong and Cao, 1987;
Feng et al., 2002).
The major breakthrough is the treatment of terminals cancer with new
approach of cell treatment. The idea is to exploit a subset of T-cells called
tumor-infiltrating lymphocytes (TILs) found deep inside cancerous tissue.
These killer T-cells attack dividing cells and provide a natural protection
against cancer (Rosenberg et al., 1993).
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Farag et al
Tumor-infiltrating lymphocytes (TILs) could be grown in vitro in a
medium containing interleukin-2 in clinical trials. Patients with metastatic
malignant melanomas were treated with interleukin-2 plus the adoptive
transfer of autologous (TILs), (Rosenberg et al., 1988).
The aim of the present work is to study the possible effects of the magic
fruit extract on the cancer cells directly or indirectly through the activation of
the TILs that normally inhibit the growth and proliferation of the cancer cells.
PATIENTS AND METHODS:
Patients: The present study has been conducted on a total of 18 female
patients, all with breast cancer taken from the outpatient clinics of the NCI of
Egypt.
Tumor tissues were obtained under the supervision of surgical and
pathological oncologists, at NCI, Cairo University, during the period from
January 2007 to August 2007.
The patients were diagnosed according to the protocols adopted at the
Surgical and Medical Oncology Departments, NCI, Egypt. They were
subjected to clinical and laboratory investigations such as liver function tests,
kidney function tests, chest X-ray, abdominal pelvic ultrasound (US), CT
scanning and isotope bone scanning.
The patients selected for our study showed the following:
-CT was free.
- Bone scan was free.
-Liver function and kidney function tests were normal.
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Chemicals and reagents:
-RPMI 1640, fetal calf serum and antibiotics (5000u penicillin and 5000 μg
streptomycin) were purchased from Sigma, USA.
-Ficoll for separating mononuclear leukocytes solution from Biochem, Kg,
Leonoemstr, Berlin.
-Enzymes and cytokines
-Collagenase - Hyaluoronidase and DNase from MP Biomedicals, France.
-Interleukin-2 human (IL-2) from Roche Diagnostic GMbH.
Preparation and Isolation of TILs
The cells were isolated according to the protocol of C ochet et
al.(1998) with some modification (Cochet et al., 1998). The cells were
subjected to activation using IL-2 (200 U/ml).
1-Freshly excised tumor tissue was washed twice with RPMI 1640 medium.
2-The tumor tissue was divided into two pieces, one for culturing and the
other for pathology.
3-The part of the tumor for the culture was minced with a scalpel into 3-5 mm
pieces.
4-This preparation was suspended into 20ml RPMI 1640 medium in culture
dish containing the following :a- DNase (1800 units)
b-Collagenase.
c-Hyaluronidase (65 units)
5-The preparation was incubated at 37 ○C for 1-2 hr with shaking.
6-The resulting cell suspension was filtered through a mesh of 380m sieve.
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Farag et al
7-The cells were washed twice using ex-vivo 15 medium.
8-Then the cells were re-suspended at conc. of 3 ×105 cells/ml in ex-vivo 15
medium with IL-2 addition at conc. of 200/ml.
9-Then the cells were transferred to 75 cm² tissue culture flasks.
10-The cells were incubated at 37○C in 5% CO2. The medium was replaced
once or twice a week.
Preparation of the magic fruit extract
The following steps were used in the preparation of the magic fruit
extract:
1-Fresh fruits were heated till boiling for 10 minutes.
2-The seeds were removed after cooling of the boiled fruits.
3-The fleshy parts of the fruit were collected and lyophilized until dryness.
4-The powdered extract was obtained and kept in dry cooled place until use.
5- 0.05 g of the powder were dissolved in 100ml of the culture medium.
Proliferation and differentiation assays:
Cell density was determined by daily counting using a hemocytometer
and cell viability test was determined by trypan blue exclusion.
Enzyme Immunoassay for interferon gamma in the cell culture:
Cell culture supernatants were centrifuged to remove any particulate
matter and assayed immediately or stored at -20 ○C until being assayed. The
IFN-gamma level was assayed using a sandwich enzyme immunoassay which
measures the free form of the cytokine. Mouse monoclonal antibodies
generated against human IFN- were used to capture human IFN- in the
sample. Simultaneously, rabbit anti-human IFN- polyclonal antibodies were
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used to detect IFN- in the sample. The reaction was visualized using goat-antrabbit alkaline phosphatase conjugate and ensuring chromogenic substrate
reaction. The amount of IFN- (pg/ml) detected was compared to a standard
curve.
Statistical analysis
Data were expressed as mean ± SE of mean. Mann-Whitney U-test was
used for non-normally distributed data. Paired t-test was used to compare the
mean level of IFN-gamma and the cell count before and after treatment. Pvalue less than 0.05 was significant.
RESULTS
Clinicopathological data of the patients:
Table 1 shows the mean, median and range of the age and the main
clinicopathological data of the patients including the tumor size, tumor grades
and stages as well as the lymph node status.
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Table 1: Clinicopathological data of the patients
No (%)
Age (yrs)
Mean ± SD
Median
range
48.88 ± 8.43
48
37-70
> 5cm
< 5cm
7(38.89 %)
11(61.11%)
II
III
12(66.67%)
6(33.33%)
2
3
12(66.67%)
6(33.33%)
Positive
negative
8 (44.44 %)
10 (55.56 %)
Tumor size
Tumor grades
Tumor stages
Lymph nodes
Effect of treatment by the magic fruit extract on the cell count and IFNlevel:
Table 2. shows the mean value of the cell count before and after
treatment of the cells by the magic fruit extract. The difference between the
cell counts before (Figs 1 & 2) and after treatment (Figs 3 & 4) is highly
significant (p< 0.001) indicating an excellent effect of the extract on
increasing the number of cells after treatment. The mean value of IFN- after
treatment was also highly significantly more than that before treatment (p<
0.001).
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Table 2: Effect of treatment of the cells by the magic fruit extract on the cell
count and IFN-level .
Cell count
IFN-
(pg/ml)
Before treatment
After treatment
p-value
Before treatment
After treatment
p-value
Mean ± SD
Median
range
5227.78 ± 3376.13
12694.44 ± 6824.24
< 0.001
10.42 ± 1.18
29.37 ± 12.65
< 0.001
4925
13700
1100 - 13800
4150 - 24800
10.25
28.40
3.9 - 12.8
15.3 - 56
Correlations between the IFN- level and the cell count:
Table 3 shows that no significant correlation was found between the IFN-
level and the cell count before and after treatment with the magic fruit extract.
Table 3:.Correlation between the IFN- level and the cell count before and
after treatment by the magic fruit extract.
Cell
count
IFNBefore treatment
r = -0.11, p = 0.963
r = -0.333, p = 0.175
Before treatment
After treatment
After treatment
r = 0.178, p = 0.477
r = -0.161, p = 0.525
Association between the clinicopathological data, the cell count and IFN level
Table 4 shows that no association was found between the
clinicopathological data, including the tumor size, grades and stage as well as
the lymph node status, and the cell count. With respect to the IFN- level, the
tumor size only was associated with IFN- level where patients with tumor
size > 5cm recorded significantly higher IFN- mean level than those with
tumor size < 5cm (p = 0.01).
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Table 4.:Association between the clinicopathological data, the cell count and
the IFNcell count
Tumor size
> 5cm (n = 7)
< 5cm (n = 11)
p-value
Tumor grades
II (n = 12)
III (n = 6)
p-value
Tumor stages
2 (n = 12)
3 (n = 6)
p-value
Lymph nodes
Positive (n = 8)
Negative (n
=10)
p-value
IFN- level
Before
After
treatment
treatment
Before
treatment
After
treatment
4900 ± 4373.69
5436.36 ± 2788.64
0.497
9435.71 ± 7564.18
14768.18 ± 5713.85
0.104
11.3 ± 1.16
9.87 ± 0.84
0.01
33.74 ± 13.64
25.59 ± 11.79
0.211
5870.83 ± 3635.46
3941.67 ± 2588.52
0.335
13945.83 ± 6372.29
10191.67 ± 7598.05
0.334
10.36 ± 1.02
10.55 ± 1.57
0.964
28.70 ± 11.55
30.71 ± 15.71
0.965
5733.33 ± 3770.29
4820.0 ± 2078.64
0.879
14054.16 ± 7535.53
11120.0 ± 3910.81
0.442
10.31 ± 1.29
10.44 ± 0.91
0.574
29.43 ± 13.83
25.76 ± 7.76
0.959
5156.25 ± 4156.35
5285.0 ± 2846.51
0.762
13462.05 ± 7770.72
12080.0 ± 6329.04
0.829
10.23 ± 1.45
10.58 ± 0.97
0.408
30.04 ± 14.43
28.84 ± 11.81
0.829
DISCUSSION
Carcinoma of the breast is the most prevalent cancer among Egyptian
women as it constitutes 29% of the NCI cases. However, early detection is the
key to improve prognosis of breast cancer. Many studies emphasized that in
Egypt, the disease is usually diagnosed at an advanced stage (Boulos et al.,
2005).
Although the use of surgery, chemotherapy and radiotherapy have led to
increasing the survival rates for patients with the breast cancer, there are still
problems of the marked side effects of the previously mentioned treatments in
addition to cancer metastasis that sometimes occurs among those patients
(Hwu and Rosenberg, 1994; Leong et al., 2006).
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In a trial to solve these problems, additional therapy could be
recommended. Therefore, the present work was designed to introduce
biological therapy using tumor infiltrating lymphocytes obtained from
malignant breast tissues, mixed with magic fruit extract and studying the
possible effect of the extract on the activity of TILs. The idea of using these
immune cells is to get rid of the remaining cancer cells that are responsible
for recurrence and metastasis and in addition to minimize dramatic
cytotoxicity of the therapy that is used nowadays.
It has been shown that the magic fruit was used as a useful therapy
against some diseases such as hypertonia, nephritis, nervous diseases and
others. In Chinese medicine, the magic fruit was found to improve the liver
function and showed a positive effect on the liver, helping the patients to
easily recover from hepatitis and cirrhosis. The anti-cancer activity of
Zizyphus jujube Mill and its underlining mechanisms of action in human
hepatoma cells (HepG2) was studied by Xuedan et al., (2007). The authors
found that the extract of Z. jujube decreased the viability of the cells.
In modern Chinese medicine, the extract is used to tone the spleen and
stomach, to treat shortness of breathing and severe emotional upset and
debility caused by nervous conditions. It was used as an antidote to poison
(Xuedan et al., 2007). In a very recent study, Shen et al., (2009) found that
the extract of Ziziphus jujube exerted some anti-oxidative activities that
caused protective effects on carbon tetrachloride-induced hepatic injury in
mice.
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The present work represents the first trial carried out in Egypt to show
the effects of the extract on the cell viability and cell number as well as the
level of IFN-. The results showed some expected observations concerning
the level of IFN- in the medium of the cell culture. The highly significant
increase in IFN- level after treatment offers a highly promising result since
IFN- is known with its crucial role in inhibiting the maturation of cancer
cells and so the increase in its level in the culture medium is a good indicator
of using the extract in treatment of breast cancer and may be prolonged to
treat other types of cancer. IFN-is one of the well known cytokines. Its
function is to up-regulate T-cell activation markers. IFN- is produced by
both macrophages and T-helper cells. (Shigeoka et al., 2003). Similar results
of increasing of IFN-was found by El-Housseini et al., (2006) due to
treatment of leukemic cells by some differentiating agents
The results also showed that the number of TILs has been significantly
increased after treatment by the extract than before treatment indicating the
possibility of using the extract as an activator of TILs before using them in
biological cancer therapy.
In conclusion, the present work casted some light on the probability of
using the magic fruit extract in the biological therapy of breast cancer through
the activation of TILs and increasing the level of IFN- in the culture media.
Future studies are recommended to examine the possibility of using the
extract in the treatment of other types of cancer.
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Figure.(1). Tumor infiltrating lymphocytes in
culture after treatment. (X 100).
Figure.(2) Tumor infiltrating lymphocytes in
culture before treatment. (X 400).
Figure.(3).Tumor infiltrating lymphocytes in
culture after treatment. (X 100). Note
the more increased number compared
to figure 1.
Figure.(4) Tumor infiltrating lymphocytes in
culture after treatment. (X 400). Note
the more increased number compared
to figure 2.
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REFERENCES
Boulos S, Gadallah M, Neguib S, Essam E & Youssef A et al.(2005): Breast
screening in the emerging world: High prevalence of breast
cancer in Cairo. Breast, 14(5):340-346.
Cochet, Q., Teilaud, J. L. & Sautes, C. (1998): Immunological techniques
made easy John Wiley & Sons Ltd., pp, 4 - 40.
El-Housseini, ME, Amer, IR Z & Hussein, TD. (2006): Effect of
differentiating agents on interferon- (IFN-level in leukemic
cells propagated ex-vivo. J. Egyp. Nat. Cancer Inst., 18(4):
283-291.
Feng ZY, Guo DW, Su S, Zhao H & Zheng XX.(2002): Sedative and
anticonvulsant effect of jujuboside A. J Zhejiang Univ
(Medical Sci), 31: 103-106.
Hong G, Cao B. (1987): Advances in research on the seed of Zizyphus
spinosa Hu. Zhong Yao Tong Bao, 12: 51-53.
Hwu, P & Rosenberg SA.(1994): The use of gene modified tumor-infiltrating
lymphocytes for cancer therapy. Annals of the New York
Academy of Sciences, 716:188-99.
Kristen, M., Drescher, L. & Henry T. (2005): Tumor infiltrating lymphocytes
(TILs): Lessons learned in 30 years of study. Clin. Appl.
Immunol. Rev., 5: 149-166.
Leong PP, Mohammad RI, Ibrahim N, Abdalla NH, Davis WC and Scow HF.
(2006): Phenotyping of lymphocytes expressing regulatory and
effector markers in infiltrating ductal carcinoma of the breast.
Immunol. Letters, 102(2):229-236.
Rosenberg SA, Lotze MT & Yang TC et al.(1993): Prospective randomized
trial of high-dose interleukin-2 alone or in conjunction with
lymphokine-activated killer cells for the treatment of patients
with advanced cancer. J. Natl. Cancer Inst., 85: 622-632.
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Rosenberg SA, Packard BS & Arebersold PM et al. (1988): Use of tumor
infiltrating lymphocytes and interleukin-2 in
the
immunotherapy of patients with metastatic melanoma, a
preliminary report. N. Engl. J. Med., 319:1676-1685.
Shen X, Tang Y, Yang R, Yu L, Fang T & Duan J. (2009): The protective
effect of Ziziphus jujube fruit on carbon tetrachloride-induced
hepatic injury in mice by anti-oxidative activities. J
Ethnopharmacology, 122(3):555-560.
Shigeoka A, Chin T, Windle M, Wilson J, Pallares D, Ballow M. (2003):
Delayed-type hypersensitivity. J. Med., 4:214-225.
Topalian, S. I., Muul, L. M., Solomon, D. Z & Rosenberg, S .A.
(1989):.Expansion of human tumor infiltrating lymphocytes for
use in immunotherapy trials. J. Immunological Methods, 102:
127 – 141.
Whiteside T. I. & Parmiani, G (1994):.Tumor infiltrating lymphocytes, their
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Immunother. 39: 15-21.
Xuedan H, Akiko KY, Toshio N, Opare KD, Tadayoshi H & Isao MY.
(2007): Mechanism of the anti-cancer activity of Zizyphus
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‫‪Farag et al‬‬
‫دور مستخلصات نبات السدر في السيطرة علي خاليا السرطان‬
‫المنزرعة خارج الجسم‬
‫رشوان محمد فرج (‪ - )1‬شادية عبد الحميد فتحي‬
‫(‪)3‬‬
‫مفيد محمد أحمد سليم‬
‫‪ )1‬كلية الطب‪ ،‬جامعة عين شمس‪ )2 .‬كلية العلوم‪ ،‬جامعة عين شمس‪ )3 .‬قسم بيولوجيا األورام‪،‬‬
‫المعهد القومي لألورام‪ ،‬جامعة القاهرة‪.‬‬
‫(‪)2‬‬
‫‪ -‬مطاوع عيسي الحسيني‬
‫[ ‪]1‬‬
‫(‪)3‬‬
‫المستخلص‬
‫الرئة‪.‬‬
‫يعد سرطان الثدي من أكثر السرطانات انتشا ار في العالم ويأتي في المرتبة الثانية بعد سرطان‬
‫ومن الوسائل المستخدمة في العالج هذه االيام الجراحة والعالج الكيميائي واالشعاعي‪ .‬وعلي‬
‫الرغم من كفاءة هذه الطرق العالجية إال أن لها الكثير من االثار الجانبية ولذلك فان العالج الحيوي‬
‫الذي يعتمد علي تنشيط الخاليا متخللة الورم ببعض المستخلصات الطبيعية مثل نبات السدر يشكل‬
‫حال بديال للعالج التقليدي ويالشي اثاره الجانبية الضارة‪.‬‬
‫ويسمي نبات السدر بالثمرة السحرية حيث وجد له من الفوائد الكثير فهو يحسن من وظائف الكبد‬
‫والكلي ويساعد في تفاعالت االيض المنتجة للطاقة وغيرها الكثير هذا فضال عن ان نبات اسدر‬
‫مذكور في القران الكريم في سورة الواقعة‪.‬‬
‫وقد أجريت هذة الدراسة لبيان تأثير مستخلص ثمار السدر علي الخاليا متخللة الورم وكذلك‬
‫علي مستوي االنترفيرون جاما في الوسط الذي تزرع فيه الخاليا‪ .‬وقد جاءت نتائج الدراسة كاالتي‪:‬‬
‫ازداد عدد الخاليا متخللة الورم والمعالجة بالمستخلص زيادة معنوية وذات داللة احصائية عنها‬
‫قبل المعالجة‪.‬‬
‫أيضا ارتفع مستوي االنترفيرون جاما في الوسط الذي تزرع فيه الخاليا متخللة الورم بعد‬
‫المعالجة بالمستخلص زيادة معنوية وذات داللة احصائية عنه قبل المعالجة‪.‬‬
‫لم يوجد ارتباط بين مستوي االنترفيرون جاما وعدد الخاليا متخللة الورم‪.‬‬
‫لم يوجد ارتباط بين البيانات السريرية المشتملة علي حجم الورم ومراحله المختلفة وحالة الغدد‬
‫الليمفاوية من سلب او ايجاب مع اي من االنترفيرون جاما وعدد الخاليا متخللة الورم‪.‬‬
‫‪15‬‬
‫‪Vol.19, No.3, Dec. 2009‬‬
‫‪J. Environ. Sci.‬‬
‫‪Institute of Environmental Studies and Research – Ain Shams University‬‬
‫ويمكن تلخيص مضمون وتوصيات الدراسة في االتي‪:‬‬
‫زيادة عدد الخاليا وكذلك زيادة االنترفيرون جاما في الوسط تعتبر نتائج مبشرة حيث يمكن‬
‫استخدام مستخلص السدر في عالج سرطان الثدي‪ .‬واالمر يحتاج الي دراسات مستقبلية لتحديد‬
‫الجرعات واألوقات المناسبة للعالج للحصول علي افضل نتائج في معالجة مرضي سرطان الثدي‪.‬‬
‫‪Vol.19, No.3, Dec. 2009‬‬
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