Anti-inflammatory and Cancer-Preventive Immunomodulation through

Chapter 22

Anti-inflammatory and Cancer-Preventive Immunomodulation through Diet Effects of Curcumin on Τ Lymphocytes Marion Man-Ying Chan and Dunne Fong

Downloaded by CORNELL UNIV on July 29, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0547.ch022

Department of Biological Sciences and Bureau of Biological Research, Rutgers, The State University of New Jersey, Piscataway, NJ 08855-1059

Curcumin, found in turmeric and curry, is a cancer preventive anti oxidant. Amongst its many functions, it inhibits cyclooxygenase and lipoxygenase dependent metabolism of arachidonic acid to prosta glandins and hydroxyeicosatetraenoic acids (HETEs), which have immuno-modulating activities. This study investigates the effects of curcumin on the Τ cell branch of the immune system: Τ helper-1 cells (Th1) which produce interleukin-2 (IL-2), Τ helper-2 cells (Th2) which produce interleukin-4 (IL-4) and cytotoxic Τ lympho cytes (CTL) which mediate tumor cell lysis. We found that commercial grade curcumin, at 10-60 μΜ, moderately enhanced IL-4 production, although IL-2 production remained unchanged. Moreover, the development of C T L was also unaffected.

The immunocompetence of the host is one of the major defenses against cancer development. In fact, the first experiment to define the role of lymphocytes in destroying cancer cells was performed as early as the 1950s (1,2). Tumors are probably outgrowths of transformed cells that have successfully escaped destruction by the immune system. Carcinogens, such as UV-irradiation, are immune suppressive (3,4), and some tumors produce immune suppressive substances; for example, mammary tumors produce prostaglandins (5,6). Anti-tumor promoters have been identified in common food sources (7-10). They include dietary fiber, vitamins A , C and E , compounds in cruciferous vegetables, and some phenolic compounds in tea and spices. These compounds prevent cancer by various mechanisms; for example, reduction of oxygen radical formation, or enhancement of the immune response. Curcumin Curcumin (diferuloylmethane), obtained from rhizomes of the plant Curcuma longa Linn, is the major yellow pigment in turmeric and curry. It is a common spice, coloring agent, and herbal drug in Asia. The antitumorigenic effect of

0097-6156/94/0547-0222$06.00/0 © 1994 American Chemical Society

Ho et al.; Food Phytochemicals for Cancer Prevention II ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


22. CHAN AND FONG

Effects of Curcumin on Τ Lymphocytes

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curcumin is well established (9-75). It inhibits tumorigenesis in mouse skin, stomach and colon. The anti-tumorigenic effect may result from a combination of mechanisms. Curcumin is an antioxidant which reduces induction of ornithine decarboxylase activity by phorbol ester, reduces polyamine synthesis and blocks oxygen free radical formation. It inhibits cyclooxygenase-dependent metabolism of arachidonic acid to prostaglandins, and metabolism of arachidonic acid by lipoxy genase to 5-HETE and 8-HETE (9). These cyclooxygenase and lipoxygenase pro ducts have inhibitory effects on the immune system (16). Therefore, curcumin may rescue the immune system from suppression caused by tumors or tumor promoters. In this chapter, we report on the effects of commercial grade curcumin on the Τ cell branch of the immune system. This crude extract contained 70% curcumin, together with 30% demethoxycurcumin and bisdemethoxycurcumin. Τ lymphocytes are divided into Τ helper cells, cytotoxic Τ lymphocytes and Τ suppressor cells. The Τ helper cells are further divided into T h l and Th2 (17). T h l cells produce IL-2 and γ-interferon (γ-INF), and are often referred to as the inflammatory Τ cells because they mediate delayed type hypersensitivity. IL-2 is important for the generation of CTL, which are tumoricidal. Th-2 cells produce IL4, formerly called Β cell growth factor (18). It has a unique function, namely the regulation of IgGi and IgE antibody production (79). Effect of Curcumin on Lymphocyte Proliferation First we tested whether curcumin is toxic to lymphocytes. Mice were infected with the parasitic protozoan Leishmania mexicana amazonensis subcutaneously. After one week, the draining lymph nodes were excised, lymph node cells were stimulated with Leishmania, and curcumin was added at various concentrations. The cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum, together with a final concentration of 0.5% acetone to aid curcumin solubility. After 3 days, the proliferation of the lymphocytes was determined by incorporation of H-thymidine. As shown in Table I, at concentrations up to 60 μΜ, curcumin did not in hibit proliferation of lymph node cells which contain Τ and Β lymphocytes. We therefore chose to use a maximum of 60 μΜ when examining effects on Τ lympho cyte function. Only 5 μΜ curcumin (ED50) is required to inhibit arachidonic acid metabolism in vitro and TPA induced tumor promotion (77). 3

Effect of Curcumin on IL-4 Production We used the murine Leishmania infection model for testing lymphokine production because it is currently one of the few systems that clearly show dichotomy for the two branches of helper cell activity (77). After infection, in the resistant C57BL/6 mice, the T h l cells, which produce IL-2 and γ-INF, are activated; whereas in the susceptible B A L B / c J mice, the Th2 cells, which produce IL-4, are activated (20,21). Therefore, in this system, the effect of the test compounds on these two compartments of the immune system can be measured independently without interference from each other. To analyze the effect of curcumin on Th2 activity, lymphocytes were prepared from Leishmania infected B A L B / c mice. After 5 days of re-stimulation in vitro, the amount of IL-4 secreted into the culture supernatants was assayed by the IL-4 specific cell line CT-4S, developed by Dr. William Paul of the National


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FOOD PHYTOCHEMICALS II: TEAS, SPICES, AND HERBS

Institutes of Health (22). The culture supernatants were serially diluted with complete RPMI (RPMI-1640 with 10% fetal calf serum) and CT-4S cells were added to each well. After an incubation of 48 hours, proliferation was determined by measurement of H-thymidine uptake. Figure 1 is a representative experiment; relative degree of IL-4 production was determined by comparing the proliferation at a log slope of the curve. Interestingly, at 10-60 μΜ, IL-4 production was enhanced, as indicated by a 2.3-fold increase in proliferation of CT-4S cells (Table II). Therefore, at a concentration that was effective for cancer prevention, curcumin enhances immune response in vitro. With respect to cancer immunity, Golumbek et al. (23) showed that introduction of IL-4 at the tumor site induces strong tumor specific C T L response and causes activation of macrophages. In the same study, they also described systemic protection from the tumor. IL-4 is synergistic with IL-2 in enhancing the in vitro and in vivo generation of CTL (24). IL-4 can also substitute for IL-2 to activate lymphokine activated killer (LAK) cells, and enhances antigen recognition by inducing major histocompatibility complex antigen (MHC) expression (25). It induces cytolytic activity of macrophages. In fact, the IL-4 induced killing is significantly higher than that of γ-INF (76).


3

Effect of Curcumin on IL-2 Production Thl cells secrete IL-2, a Τ cell growth factor that potentiates immunity in general. In cancer immunotherapy, IL-2 has been used to culture L A K and tumor-infil trating lymphocytes (TIL) from excised tumors and has also been used directly with some degree of success, although IL-2 therapy produces severe adverse side effects (26, 27). Therefore, we investigated the effect of curcumin on IL-2 production. In contrast to Th2, T h l lymphocytes were prepared from Leishmania infected C57BL/6J mice, and the quantity of IL-2 in the culture supernatants was measured by IL-2 specific CT-2 cells from Dr. Frank Fitch of the University of Chicago (28). As shown in Table III, unlike its effect on IL-4, curcumin did not affect the production of IL-2. The proliferation index was 1.06 after addition of curcumin at concentrations that enhanced the production of IL-4. Since both CT-2 and CT-4S are derived from the same parental line C T L L 2, comparison between the IL-2 and IL-4 experiments also indicated that it is highly unlikely that curcumin affects the indicator cells. Effect of Curcumin on CTL Cytotoxic Τ lymphocytes are responsible for lysis of cancer cells and IL-2 is essential for proliferation of C T L (29-31). Therefore, we studied the effect of curcumin on cytotoxic Τ lymphocyte generation. Allogeneic C T L were generated by culturing the spleen cells from two strains of mice, C57BL/6J (H-2 ) and B A L B / c J (H-2 ), for 5 days. The CTL activity was evaluated by cytolysis of C r labeled P815 mastocytoma target cells (H-2 ). As shown in Table IV, curcumin, at 10-50 μΜ, did not affect the generation of allogeneic CTL. b

d

51

d

Curcumin as a Potential Dietary Immune Modifier The NCI has a program for the development of biological response modifiers that would modify a patient's response to fight tumors (32). Immunostimulants have


22. CHAN AND FONG


225


Table I. Toxicity of Curcumin on Lymphocytes Concentration of curcumin (μΜ) 60 30

Experiment

0

1

55±2

2 3

125 3 ±0.6

58 ± 6

52±9

65 + 12

64±3

65 ± 8

52±6

115 ± 1 0

126 + 9

124 ± 4

112+12

a

3

Counts per minute (χ 10 , mean ± S. E.) 5

Lymph node cells (1 χ 10 ) from mice that had been infected at the tail base with 4 χ 10 Leishmania promastigotes were restimulated with promastigotes (1 χ 10 ) in vitro, in 96 well plates. Curcumin was added to the cultures at the indicated concentrations. After 4 days, H-thymidine was added and the cultures were incubated for 12-16 hours. The cells were then harvested by a cell harvestor and the amount of radioactivity incorporated was determined by liquid scintillation counting. 6

4

3

30000 4>

Dilution of supernatant (1/n) Figure 1. Example of an IL-4 assay. Culture supernatants were serially diluted at 1:2 ratio in 96 well plates, then equal volumes of thoroughly washed CT-4S cells, which had been grown in recombinant IL-4, were added at 5 χ 10 cells/ ml. The plates were incubated overnight, and then H-thymidine was added. After another 12-14 hours, the cells were harvested by a cell harvestor and the samples were prepared for liquid scintillation counting. 4

3



226

Table Π. Effect of Curcumin on IL-4 Production Experiment


1

2

3

4

5

6

Curcumin (μΜ)

Proliferation index

CPM a

0 60

8163 26865

0 30 60

12266 29009 29196

2.1 2.4

0 30 60

2416 4148 3249

1.7 1.3

0 10 60

13577 22521 19621

1.7 1.5

0 10 50

3025 12266 13034

4.2 4.3

0 10 50

27879 32940 37839

1.2 1.4

3.3

Average Range a b c

b

2.3±1.2 1.1-3.5

C

Counts per minute Proliferation index: C P M with curcumin/CPM without curcumin Mean±S.E.

Lymph node cells from Leishmania infected C57BL/6J mice were restimulated in vitro in the presence of the indicated concentrations of curcumin as described in Table I. After 5 days of incubation at 37°C, the supernatants were collected and tested for IL-4 as described in Figure 1.


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Table III. Effect of Curcumin on IL-2 Production Experiment 1


2

3

4

Curcumin (μΜ)

CPM

Proliferation index a

0 60

26672 22150

0 30 60

10384 12052 19509

1.2 1.9

0 10 30 60

65558 67841 67974 34332

1.0 1.0 0.52

0 10 30 60

4899 5384 4266 4724

1.08 0.87 0.96

0 10 50

10931 10677 13059

0.97 1.2

0 10 50

5772 6749 6169

1.2 1.1

0.83

Average Range a b

c

b

1.06±0.30 0.76-1.36

c

Counts per minute Proliferation index: C P M with curcumin/CPM without curcumin Mean±S.E.

Lymph node cells from Leishmania infected B A L B / c J mice were restimulated in vitro in the presence of the indicated concentrations of curcumin, as described in Table I. After 2 days of incubation at 37°C, the supernatants were collected and tested for IL-2 in a manner similar to that described in Figure 1, except CT-2 cells were used.




228

been successful in clinical trials. For example, bacillus Calmette Guerin (BCG) from mycobacterium, which enhances activation of Τ lymphocytes and macrophages, has protected patients against bladder carcinoma, acute nonlymphocytic leukemia, and lung cancer in clinical trials (33, 34). Levamisole is another immune enhancer which has been effectively used against mammary, hepatic, and colon carcinomas; it stimulates IL-2 synthesis, γ-INF production, and activates polymorphonuclear leukocytes and macrophages (34-36). In this report, we identified curcumin, an antitumorigenie compound, as another potential immuno-modulatory compound. Since it is present in commonly comsumed food, this information will be useful for implementing cancer preventive diets. In addition, this information shows that avoiding curcumin in the diet may be helpful in some immune diseases, such as allergies and autoimmune diseases, because consumption of curcumin may enhance IL-4 and thus aggravate the symptoms.

Table IV. Effect of Curcumin on Cytotoxic Τ Lymphocyte Generation Concentration of curcumin (μΜ) 10 50

Experiment

0

1

57

2 3 Average a b

65

76

66

56

57

74

72

84

64 ± 8.3

72 ± 1 4

a

65±8

b

Percent cytolysis Mean±S.E.

Allogeneic C T L were generated as described in the text. The target cells were labeled with 0.1 mCi of Na CrC>4, for one hour at 37°C and washed to remove unbound label. The spleen cell preparation and target cells were then mixed. After incubation, an aliquot of the supernatant was removed for determination of the amount of radioactivity released. The percentage of C r released from the target cells due to C T L activity was calculated by the formula: Percent specific lysis = 100 χ ( C r released - spontaneous C r released) / (maximum Cr released - spontaneous C r released). Maximum C r release was determined by complete lysis of labeled target cells in 0.5 Ν HC1. 51

5 1

51

5 1

5 1

5 1

5 1

Acknowledgements The authors thank Professors A . H . Conney and M-T. Huang for advice and supply of curcumin, and Professors W. E. Paul and F. Fitch for cell lines. This work was supported in part by grant 910024 from the World Health Organization to M.M.C., and grants BC695 from the American Cancer Society and CA49359 from the National Institutes of Health to D.F.


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Anti-inflammatory and Cancer-Preventive Immunomodulation through

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