Am J Clin Oncol (CCT) 26(4): 416–421, 2003.

© 2003 Lippincott Williams & Wilkins, Inc., Philadelphia

Prognostic Value of Serum IL-18 and Nitric Oxide Activity in Breast Cancer Patients at Operable Stage Nazan Günel, M.D., Ug˘ur Cos¸kun, M.D., Banu Sancak, M.D., Og˘uz Hasdemir, M.D., Mustafa Sare, M.D., Orhan Bayram, M.D., Gokhan Celenkoglu, M.D., and Secil Ozkan, M.D.

enhancing natural killer (NK) cell cytotoxicity.4,5 Moreover, IL-18 has been shown to have potent antitumor effects that are mediated by reduction of tumorigenesis,6 inhibition of angiogenesis,7 and induction of apoptosis8 in tumor cells. Recently, it has been suggested that serum IL-18 may have prognostic importance in some cancer types including colon,9 gastric carcinoma,10 and malignant lymphoma.11 Nitric oxide (NO) is a potent biologic molecule of physiologic functions such as vasodilatation, neurotransmission, host defense and carcinogenesis.12–14 NO plays a complex and sometimes opposite role in tumorigenesis. The association between increased NO activity and tumor progression is proposed by some studies,15,16 whereas others showed a positive correlation with decreased NO activity.17,18 Some specific functions of NO in tumorigenesis are thought to be related to its concentration in tumor tissue and its interactions with other molecules including IL-18, IFN-␥, and tumor necrosis factor-␣.19,20 It has been shown that IL-18 caused marked increases in serum NO levels in animal models.19 Recently, it has been demonstrated that increased NO activity negatively correlates with histologic grade and lymph node status and positively correlates with estrogen receptor (ER) expression in breast carcinoma.21 In the present study, our aim was to investigate the role of serum IL-18 and NO activity as a prognostic predictor in patients with breast cancer.

Interleukin-18 (IL-18) is a multifunctional cytokine that was previously termed interferon-␥-inducing factor. It has been suggested that serum IL-18 level may be used as a prognostic factor in some cancer types. Nitric oxide is a potent biologic molecule involved in the pathogenesis of cancer. In this study, we measured serum IL-18 and nitrate ⫹ nitrite levels in 56 patients with nonmetastatic breast cancer and 14 control subjects. Serum IL-18* and nitrate ⫹ nitrite** levels were significantly higher in patients with breast cancer when compared to the control subjects (*p ⬍ 0.05, **p ⬍ 0.001). Serum IL-18 levels were significantly higher in patients whose tumor size was greater than or equal to 5 cm when compared to patients whose tumor size was less than or equal to 2 cm (p ⬍ 0.05). Patients who were axillary lymph node negative (ALN) had lower serum IL-18 levels when compared to patients with positive ALN (p ⬍ 0.001). Serum IL-18 levels were significantly higher in patients with stage IIB or IIIA when compared to patients with stage I or IIA (p ⬍ 0.05). There was no significant difference in serum nitrate ⫹ nitrite levels in terms of age, tumor stage, estrogen receptor, and menopausal and ALN status (p ⬎ 0.05). In conclusion, serum IL-18 level may be a useful marker to predict prognosis of patients with breast cancer in complete remission after surgery. Long-term follow-up is required to clarify this hypothesis. Key Words: IL-18 —Nitric oxide—Breast cancer—Complete remission—Prognosis.

Interleukin-18 (IL-18) is a novel cytokine that was initially termed as interferon-␥ (IFN-␥)-inducing factor.1 IL-18 is produced by macrophages, Kupffer cells, keratinocytes, intestinal epithelial cells, osteoblasts, and adrenal cortex cells as a biologically inactive form that requires cleavage with caspase-1 to become active.2,3 Human IL-18 has multiple biologic activities including augmenting IFN-␥ and granulocyte-macrophage colonystimulating factor production in mononuclear cells and

MATERIALS AND METHODS Fifty-six patients with breast cancer in complete remission after surgery and after adjuvant chemotherapy and 14 control subjects were investigated. All patients were histologically confirmed with breast cancer TNM (tumor-node-metastasis) staging.22 Informed consent was obtained from all patients and control subjects. To eliminate the influence of other diseases, we excluded patients with infectious diseases, hypertension, diabetes mellitus, lung disease, renal failure, and synchronous secondary malignancies. Patients with metastatic diseases and under hormone therapy were also excluded. Median age was 50.5 years (range: 29 –73 years) and 46.5 years (range: 31– 68 years) in patients and control subjects, respectively. All patients

From the Departments of Medical Oncology (N.G., U.C., G.C.), Biochemistry (B.S.), and Surgery (O.B., M.S.), Public Health (S.O.), and Gazi University Medical School, Department of Surgery (O.H.), SSK Ankara Hospital, Ankara, Turkey. Address correspondence and reprint requests to Prof. Nazan Gu¨nel, Gati Universiti, TIP Fakultesi Hastanesi, Medikal Onkoloji, Besevlar/ Ankana, Turkey.

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IL-18 AND NITRIC OXIDE ACTIVITY IN BREAST CANCER were treated with anthracycline-based chemotherapy as adjuvant setting. Four or more patients who were axillary lymph node (ALN) positive also received taxanes. Median follow-up time was 16 months (range: 6 –112 months) after surgery. Serum samples were obtained from patients at least 4 weeks after chemotherapy between 9:00 and 11:00 AM after overnight fasting. Samples were stored at ⫺30°C until analysis.

Statistical Analysis

The results were presented as mean ⫾ SD. Mann Whitney U test and Pearson’s correlation analysis were used in statistical analysis. p values less than 0.05 were accepted as significant.

IL-18 Measurement Enzyme-linked immunosorbent assay (ELISA) for determination of IL-18 in serum was performed according to a previously described method.23 Immediately after blood sampling, serum was obtained by centrifugation at 800g at 4°C for 15 minutes. The IL-18 level in each sample was determined by using commercially available ELISA kits (BioSource International human IL-18 Colorimetric solid phase Sandwich ELISA, San Francisco, CA, U.S.A.). Sensitivity was determined by assaying serially diluted hIL-18 Calibrator. The mean absorbance plus 2 SD for Calibrator diluted to 6.25 pg/ml was lower than the mean absorbance minus 2 SD for Calibrator diluted to 12.5 pg/ml. The minimal detectable dose is therefore 12.5 pg/ml.

Nitrite and Nitrate Measurement Nitrite was measured by using the Griess reaction and the results are given as micromoles per liter.24 Nitrate was measured by using the enzymatic one-step assay with nitrate reductase.25 The method was based on the reduction of nitrate to nitrite by nitrate reductase in the presence of ␤-NADPH. We equilibrated tubes at 25°C containing 250 ␮l 100 mmol/l potassium phosphate buffer (p H 7.5) and 50 ␮l 12 mmol/l ␤-NADPH with 100 ␮l sample. To start the enzymatic reaction, we added 40 ␮l 500 U/l nitrate reductase. We incubated the tubes in the dark for 45 minutes. The concomitant oxidation of ␤-NADPH was monitored by the decrease in absorbance at 340 nm. The method of standard addition was used to minimize the effect of interfering substances via serum. The results were expressed as micromoles per liter. We also used samples with internal standard, serum blanks, and reagent blank.

CEA and CA 15-3 Measurement CA 15-3 was determined by a commercial enzyme immunoassay adapted to an ES-700 analyzer as previously reported.26

RESULTS There was no difference between patients with breast cancer and control subjects in terms of age (Table-1). Serum IL-18 and nitrate ⫹ nitrite levels were significantly higher in patients with breast cancer when compared to the control subjects (IL-18: 344.8 ⫾ 197.2 vs. 195.9 ⫾ 67.9 pg/ml, p ⬍ 0.05; nitrate ⫹ nitrite: 324.6 ⫾ 132.9 vs. 56.5 ⫾ 24.3 ␮mol/l, p ⬍ 0.001) ( Figs. 1 and 2). Serum IL-18 levels were significantly higher in the patients whose tumor size were ⱖ 5 cm (451.5 ⫾ 247.2 pg/ml) when compared to the patients whose tumor size were 2 cm or less (236.2 ⫾ 76.8 pg/ml, p ⬍ 0.05) (Table

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1, Fig. 3). Serum IL-18 levels were significantly lower in the control subjects (195.9 ⫾ 67.9 pg/ml) when compared to the patients whose tumor size was between 2 and 5 cm (345.6 ⫾ 190.4 pg/ml, p ⬍ 0.05) or 5 cm or more (451.5 ⫾ 247.2 pg/ml, p ⬍ 0.001). However, there was no difference between the patients whose tumor size were 2 cm or less and control subjects (p ⬎ 0.05). There was no difference in serum IL-18 levels between the patients whose tumor size was between 2 and 5 cm and 5 cm or more (p ⬎ 0.05) (Fig. 3). No significant difference was found in serum nitrate ⫹ nitrite levels in patients with breast cancer in terms of tumor size (p ⬎ 0.05) (Table-1). ALN negative patients (237.7 ⫾ 91.7 pg/ml) had lower serum IL-18 levels when compared to the patients who had 1 to 3 (440.1 ⫾ 273.4 pg/ml) or 4 or more positive ALN (434.7 ⫾ 120.5 pg/ml, p ⬍ 0.05) (Table-1, Fig. 4). However, there was no difference between the patients who had 1 to 3 and 4 or more positive ALN (p ⬎ 0.05). Serum IL-18 levels were significantly higher in the patients who had 1 to 3 or 4 or more positive ALN when compared to the control subjects (p ⬍ 0.001). However, no significant difference was found between ALN negative patients and control subjects (p ⬎ 0.05) (Fig. 4). There was no significant difference in serum nitrate ⫹ nitrite levels in patients with breast cancer in terms of ALN status (p ⬎ 0.05) (Table 1). There was no significant difference in serum IL-18 and nitrate ⫹ nitrite levels in terms of ER status (p ⬎ 0.05) (Table 1). Serum IL-18 levels were significantly higher in the patients with stage IIB (406.3 ⫾ 198.8 pg/ml) or IIIA (552.0 ⫾ 266.9 pg/ml) diseases when compared to the patients with stage I (193.8 ⫾ 57.1 pg/ml) or IIA (249.8 ⫾ 76.1 pg/ml) diseases (p ⬍ 0.05) (Fig. 5). Serum IL-18 levels were significantly higher in the patients with stage-IIA*, IIB**, or IIIA** diseases when compared to the control subjects (*p ⬍ 0.05, **p ⬍ 0.001). However, no significant difference was found between the patients with stage I disease and control subjects (p ⬎ 0.05). There was no significant difference in serum IL-18 levels between the patients with stage I and IIA diseases and between the stage IIB and IIIA diseases (p ⬎ 0.05) (Fig. 5). No significant difference was found in serum nitrate ⫹ nitrite levels in patients with breast cancer in terms of disease stage (p ⬎ 0.05) (Table 1). There was no significant difference in serum IL-18 levels in terms of tumor grade (p ⬎ 0.05) (Table 1). Serum nitrate ⫹ nitrite levels were lower in the patients with grade III (294.5 ⫾ 124.9 ␮mol/l) disease when compared to the patients with grade I (335.4 ⫾ 140.8 ␮mol/l) and II (331.4 ⫾ 138.6 ␮mol/l) diseases, but this was not statistically significant (p ⬎ 0.05). There was no significant difference in serum IL-18 and nitrate ⫹ nitrite levels in patients with breast cancer in terms of menopausal status (p ⬎ 0.05) (Table 1). There was a positive correlation between serum IL-18 and nitrate ⫹ nitrite levels (r ⫽ 0.238, p ⬎ 0.05). Am J Clin Oncol (CCT), Vol. 26, No. 4, 2003

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N. GÜNEL ET AL. TABLE 1. Patient characteristics

All patients Control subjects Tumor size (cm) Tm ⱕ 2 Tm ⫽ 2–5 Tm ⱖ 5 ALN (⫺) 1–3 (⫹) 4 and 1 (⫹) ALN (⫺) (⫹) Estrogen receptor (⫺) Mild (⫹) Moderate (⫹) Estrogen receptor (⫺) (⫹) Stage I IIA IIB IIIA Stage I ⫹ IIA IIB ⫹ IIIA Grade 1 2 3 Menopausal status PrePost-

n

Age (median) (y)

IL-18 (pg/ml)

Nitrite ⫹ nitrate (␮mol/l)

CEA (ng/ml)

CA15-3 (U/ml)

56 14

50.5 (29–73) 46.5 (31–68)

344.8 ⫾ 197.2 195.9 ⫾ 67.9

324.6 ⫾ 132.9 56.5 ⫾ 24.3

2.1 ⫾ 1.0 —

26.6 ⫾ 10.7 —

12 32 12

51.5 (38–73) 53.0 (29–70) 44.5 (34–72)

236.2 ⫾ 76.8 345.6 ⫾ 190.4 451.5 ⫾ 247.2

362.5 ⫾ 171.5 318.5 ⫾ 117.9 302.7 ⫾ 142.2

1.9 ⫾ 0.7 2.0 ⫾ 1.0 2.4 ⫾ 1.4

23.7 ⫾ 9.4 27.9 ⫾ 10.8 25.9 ⫾ 12.1

26 17 13

50.0 (32–73) 51.0 (29–70) 43.0 (34–72)

237.7 ⫾ 91.7 440.1 ⫾ 273.4 434.7 ⫾ 120.5

292.2 ⫾ 102.0 389.6 ⫾ 158.5 304.2 ⫾ 139.8

1.9 ⫾ 1.0 2.2 ⫾ 1.0 2.3 ⫾ 1.3

25.6 ⫾ 10.0 26.5 ⫾ 11.0 28.7 ⫾ 12.2

26 30

50.0 (32–73) 50.5 (29–72)

237.7 ⫾ 91.7 437.7 ⫾ 217.4

292.2 ⫾ 102.0 352.6 ⫾ 152.0

1.9 ⫾ 1.0 2.2 ⫾ 1.1

25.6 ⫾ 10.0 27.5 ⫾ 11.4

14 20 22

43.0 (29–61) 50.5 (34–65) 52.0 (32–73)

397.9 ⫾ 272.4 337.3 ⫾ 153.4 317.9 ⫾ 178.7

280.5 ⫾ 124.3 329.7 ⫾ 107.6 347.9 ⫾ 160.4

2.0 ⫾ 1.3 2.5 ⫾ 1.0 1.8 ⫾ 0.9

28.8 ⫾ 10.7 28.5 ⫾ 11.2 23.4 ⫾ 10.0

14 42

43.0 (29–61) 53.5 (32–73)

397.9 ⫾ 272.4 327.2 ⫾ 165.4

280.5 ⫾ 124.3 339.3 ⫾ 136.5

2.0 ⫾ 1.3 2.1 ⫾ 1.0

28.8 ⫾ 10.7 25.9 ⫾ 10.8

7 19 23 7

52.0 (44–73) 49.0 (32–65) 51.0 (29–70) 42.0 (34–72)

193.8 ⫾ 57.1 249.8 ⫾ 76.1 406.3 ⫾ 198.8 552.0 ⫾ 266.9

278.9 ⫾ 80.0 355.3 ⫾ 148.3 310.2 ⫾ 136.5 334.2 ⫾ 140.8

1.7 ⫾ 0.5 2.0 ⫾ 1.0 2.2 ⫾ 0.9 2.3 ⫾ 1.7

23.4 ⫾ 9.1 25.6 ⫾ 10.4 28.8 ⫾ 10.8 25.1 ⫾ 13.5

26 30

50.5 (32–73) 50.0 (29–72)

234.7 ⫾ 74.8 440.3 ⫾ 220.5

334.7 ⫾ 136.3 315.8 ⫾ 135.4

1.9 ⫾ 0.9 2.3 ⫾ 1.1

25.0 ⫾ 9.9 27.9 ⫾ 11.4

15 29 12

49.0 (34–69) 51.0 (32–73) 45.0 (29–71)

351.2 ⫾ 207.2 348.5 ⫾ 138.3 338.9 ⫾ 279.5

335.4 ⫾ 140.8 331.4 ⫾ 138.6 294.5 ⫾ 124.9

2.1 ⫾ 1.4 2.1 ⫾ 0.9 1.9 ⫾ 1.0

25.0 ⫾ 12.4 29.7 ⫾ 10.2 19.2 ⫾ 12.6

29 27

42.3 (29–51) 63.0 (51–73)

362.8 ⫾ 178.8 325.5 ⫾ 216.9

328.1 ⫾ 163.7 320.8 ⫾ 98.0

2.3 ⫾ 1.1 1.8 ⫾ 1.0

28.0 ⫾ 10.9 25.0 ⫾ 10.5

ALN, axillary lymph node; Tm, tumor. Normal range of CA15–3: 7.5–53 U/ml, carcinoembryonic antigen (CEA): 0.0 –10.0 ng/ml.

FIG. 1. Serum IL-18 levels in patients and control subjects.

Am J Clin Oncol (CCT), Vol. 26, No. 4, 2003

FIG. 2. Serum nitrate⫹nitrate levels in patients and control subjects.

IL-18 AND NITRIC OXIDE ACTIVITY IN BREAST CANCER

FIG. 3. Serum IL-18 levels in patients according to tumor size.

Moreover, serum IL-18 levels were also positively correlated with tumor size (r ⫽ 0.418, p ⬍ 0.05). There was a negative correlation between serum nitrate ⫹ nitrite levels and tumor grade (r ⫽ -0.577, p ⬍ 0.001). Serum IL-18 and nitrate ⫹ nitrite levels were not correlated with serum carcinoembryonic antigen and CA-15-3 levels (p ⬎ 0.05). DISCUSSION IL-18, previously termed IFN-␥-inducing factor, is an 18.3-kD multifunctional cytokine.1 In addition to its IFN-␥ inducing function, it has a variety of biologic

FIG. 4. Serum IL-18 levels in patients according to ALN status.

419

FIG. 5. Serum IL-18 levels in patients according to tumor stage.

actions including the proliferation of T cells, enhancing cytotoxic activity of natural killer (NK) cells, inducing secretion of granulocyte-macrophage colony-stimulating factor from NK and T-cells.1,4 Recently, it has been suggested that inappropriate production of IL-18 contributes to the pathogenesis of cancer and may influence the clinical outcome of patients.27 Taniguchi et al.23 reported that patients with acute and chronic leukemia showed significantly increased serum IL-18 levels compared to the control group. They suggested that serum IL-18 level could be a sensitive indicator for monitoring the therapeutic effect and the clinical course of hematologic malignancies.23 Tokubo et al.11 reported that serum IL-18 level should be considered as a new prognostic factor for patients with nonHodgkin’s lymphoma. In another study, it has been suggested that the decrease in local production of IL-18 and the subsequent impairment of immune cell activation may favor tumor growth and may be an early event in tumor progression of colon cancer.9 IL-18 production may be induced in response to the tumor cells or other factors related to tumor development, and its production is constitutive or prolonged.10,28,29 Merendino et al.30 showed that patients with breast cancer with liver and bone metastases had higher serum IL-18 levels when compared to the control group. In our study, patients with breast cancer showed significantly increased serum IL-18 levels when compared to the control subjects. All patients were in complete remission with no evidence of metastatic disease in this study. However, median follow-up time has not reached enough duration to draw a clear conclusion. Elevated serum IL-18 levels in non-MBC patients may be related to the prolongation of IL-18 induction after surgery to eliminate preexisting tumor cells and generate long-lasting immunity. Kawabata et al.10 reported that the preoperative serum Am J Clin Oncol (CCT), Vol. 26, No. 4, 2003

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N. GÜNEL ET AL.

IL-18 level may be a significant prognostic determinant in gastric cancer, and the patients in each stage who had high serum IL-18 levels also experienced poorer survival compared with patients who had lower levels. In our study, patients whose tumor sites were 5 cm or more had higher serum IL-18 levels in comparison with the patients whose tumor sizes were 2 cm or less (p ⬍ 0.05). Serum IL-18 levels were not different in patients whose tumor sizes were 2 cm or less when compared to the control subjects. In accordance with this finding, advanced stage (IIB, IIIA) and ALN(⫹) patients showed significantly higher serum IL-18 levels when compared to the ALN(⫺) and stage (I, IIA) patients. However, there was no association between serum IL-18 levels and other prognostic factors including age, ER status, and tumor grade. Higher serum IL-18 levels in patients with larger tumor size and positive ALN and thus with relatively advanced stage may reflect the increased immunologic responses at initial diagnosis. In our study, it is not possible to conclude whether this serum IL-18 level is a predictive marker for tumor relapse related with microscopic residual disease or whether it reflects the degree of immunodefense mechanism against tumor overgrowth. Long-term follow-up of these patients will make it possible to discuss much more about these hypotheses. However, we can conclude that serum IL-18 levels may a candidate as a new prognostic marker for patients with breast cancer in complete remission after surgery. It has been emphasized that the antitumor effects of IL-18 may not be limited to the activity of IFN-␥. The production of other cytokines induced by IL-18 such as tumor necrosis factor-␣, IL-1␤, IL-8, and NO might certainly be involved in the antitumor response as well.7,31 It has been demonstrated that IL-18 induces macrophages to produce NO,32 and changes in serum IFN-␥ levels were positively correlated with NO in terms of both time and levels after induction of IL-18.19 Wang et al.33 showed that IFN-␥ was induced during tumor growth and metastasis and this IFN-␥ production led to the upregulation of the NO synthetase II gene and production of NO at a higher level. Macrophages produce higher levels of NO than tumor cells or other host cells, and they are the main source of NO synthesis.34 –37 It has been shown that NO is synthesized by several cancer lines21 and solid tumors.13,38 However, its precise function in cancer biology is not yet clearly understood.21 NO may prevent tumor growth and metastasis by induction of apoptosis.39 On the other hand, increased NO activity may promote tumor growth and metastasis by causing DNA damage and inducing immunosuppression, vasodilatation, and angiogenesis.34,40 The balance between these opposite roles in tumorigenesis appear to depend on the degree of NO production in tumor tissue and cellular status of tumor cells. Increased NO concentrations may exert antiproliferative effects, and decreased concentrations may facilitate tumor growth.41,42 It has been reported that NO synthase activity was higher in invasive tumors when compared to benign Am J Clin Oncol (CCT), Vol. 26, No. 4, 2003

tumors or normal tissue in breast cancer.13 Moreover, it has been found that endothelial NO synthase was expressed in human breast cancer cell lines,43 and NO inhibited the proliferation of breast cancer cells by inducing apoptosis.43,44 In our study, serum nitrate ⫹ nitrite levels have been measured as an index of NO generation.45 All patients had higher nitrate ⫹ nitrite serum levels when compared to the control subjects. These increased nitrate ⫹ nitrite levels may be a reflection of the host defense mechanism by inducing NO synthase to prevent tumor growth and metastasis. Martin et al.41 showed that endothelial NO synthase activity was expressed by human breast tumors and that its presence negatively correlates with histologic grade and lymph node status but positively correlates with ER expression. In contrast to these findings, a positive correlation between NO synthase activity and tumor grade in breast cancer has also been reported in other studies.13,44 In the present study, negative correlation was found between serum nitrate ⫹ nitrite levels and tumor grade similar to Martin’s study. However, serum nitrate ⫹ nitrite levels were not correlated with other prognostic factors including tumor size, stage, age, ER, and menopausal status in our study. In conclusion, we showed significantly higher serum IL-18 and nitrate ⫹ nitrite levels in patients with breast cancer when compared to the control subjects. Serum IL-18 levels were higher in patients with larger tumor size, positive ALN, and advanced stage. Serum IL-18 level may be a prognostic marker for monitoring the clinical follow-up of breast cancer. To clarify this suggestion, larger studies with long-term follow-up are needed. REFERENCES 1. Okamura H, Tsutsui H, Komatsu T, et al. Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature 1995;378:88 –91. 2. Gu Y, Kuida K, Tsutsui H, et al. Activation of interferon-gamma inducing factor mediated by interleukin-1 beta converting enzyme. Science 1997;275:206 –9. 3. Takubo T, Okura H, Kumura T, et al. Human IL-18 bioactivity in hematological malignancies with highly elevated serum IL-18 levels. Acta Haematol 2000;103:162– 4. 4. Micallef MJ, Ohtsuki T, Kohno K, et al. Interferon-gamma-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferongamma production. Eur J Immunol 1996;26:1647–51. 5. Ushio S, Namba M, Okura T, et al. Cloning of the cDNA for human IFN-gamma-inducing factor, expression in Escherichia coli and studies on the biologic activities of the protein. J Immunol 1996;156:4274 –9. 6. Fukumoto H, Nishio M, Nishio K, et al. Interferon-gamma-inducing factor gene transfection into Lewis lung carcinoma cells reduces tumorigenicity in vivo. Jpn J Cancer Res 1997;88:501–5. 7. Park CC, Morel JC, Amin MA, et al. Evidence of IL-18 as a novel angiogenic mediator. J Immunol 2000;167:1644 –53. 8. Ohisuki T, Micallef MJ, Tanimoto T, et al. Interleukin-18 enhances fas ligand expression and induces apoptosis in fas-expressing human myelomonocytic KG-1 cells. Anticancer Res 1997;17: 3253– 8.

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