The Role of Interleukin-8 in the Progression of Malignant Mesothelioma

Transcription

1 Ph. D. Thesis The Role of Interleukin-8 in the Progression of Malignant Mesothelioma Gabriella Gálffy M.D. Semmelweis University, School of PhD Studies Consultant: Prof. Pál Magyar M.D. Budapest, 2001

2 Introduction Malignant mesothelioma is a relatively rare, but a very aggressive tumor that is resistant to any. It is characterized by local tumor extension without distant metastasis and invasion of surrounding tissue, such as phrenic muscle, mediastinum and pleural space including visceral and parietal pleural surfaces. Regarding that, the prognosis is poor, despite of early diagnosis and use of combination therapies such as radiation, chemotherapy and surgical treatment. In the United States, there are approximately 2000 deaths per year and 4000 newly diagnosed cases. The average survival time is about 7-9 months after diagnosis. Unfortunately, its incidence appears to be increasing. It is a relatively rare tumor, but its incidence appears to be increasing. The current annual incidence of malignant mesotheliomas is approximately 11.4/1 million in men and 2.8 / 1 million in women in USA (1). Individuals with a history of exposure to asbestos have a much greater risk of developing these neoplasms, although at least 20% of tumors may develop spontaneously. Unfortunately, there is no effective treatment for prolonging survival at this time. Several tumors produce specific autocrine growth factors which improve their growth capacity, tissue invasion and ability for metastasis. Based on experimental results, interleukin-8 (IL-8) is such a factor. IL-8 is a member of the super gene family of CXC chemokines with multifactorial effects. As a proinflammatory cytokine, it causes activation, chemotaxis, tissue penetration and degranulation of neutrophils. Similarly to Vascular Endothelial Growth Factor, Tumor Necrosis Factor-α, Fibroblast Growth Factor, IL-8 has been described as an important angiogenic factor for the development of new capillaries, in vivo and in vitro. Several immune and non-immune cells, including endothelial and mesothelial cells have been found to produce IL-8. In addition, IL-8 has also been shown to be generated by a variety of tumors such as malignant melanoma and bronchogenic carcinoma (non small cell lung cancer). It serves as an autocrine growth factor, in case of certain malignant melanoma, allowing for local tumor growth and tissue invasion. In non small cell carcinomas, however, IL-8 is not functioning as an autocrine growth factor, but regarding its angiogenic capacity, the IL-8 levels of pleural fluid and the progression of the tumors showed significant correlation. IL-8 Ab blocked the proliferative effects of IL-8 in malignant melanoma and also decreased the progression after IL-8 administration in NSCLC. In pleural fluids of malignant mesothelioma similarly to NSCLC, elevated concentrations of IL-8 were found. These levels were higher, than in lung cancer. These data along with the well known angiogenic and proliferative effects of IL-8 have led us to examine the prognostic role of IL-8 in the progression of malignant mesothelioma.

3 Aims In our current work, we aimed to investigate the role of IL-8 in the progression of malignant pleural mesothelioma. In doing this, we determined differences in IL-8 levels from human malignant mesothelioma effusions and congestive heart failure pleural fluids (CHF) as control. It was also designed to measure the level of IL-8 in human mesothelioma and mesothelial cell culture supernatants, in vitro. To determine the effect of human IL-8 protein in regulating human mesothelioma and mesothelial cell proliferation, IL-8 and anti-il-8 antibody were added to the cell cultures and the proliferative capacity was measured. After that, we also aimed to develop a "patientlike" model to assess the role of IL-8 in vivo during tumorigenesis of human malignant pleural mesothelioma. Most experimental pleural mesothelioma models, which used for testing the biologic response to therapeutic interventions and agents are not a "patient like" animal tumor model. After inhalation or inoculation of asbestos or other fibers, the tumors are not human origins and the development is long and rear. In our model, athymic nude mice were intrapleural injected by human malignant mesothelioma cells (CRL-2081) and treated with anti IL-8 Ab or control IgG. Patients and Methods IL-8 levels were measured in malignant mesothelioma and chronic heart failure pleural fluids and in supernatants of 3 different cultured malignant mesothelioma (CRL- 2081, CRL-5915 and CRL-5820) and 1 mesothelial (CRL-9444) cell lines, in vitro by an ELISA technique. Conditioned media were collected and measured at 24, 48, 72 and 96 hours of incubation. The proliferative activity after IL-8 administration was determined by thymidine ( 3 H-TdR) incorporation and also by direct cell counts after incubation with varying concentrations of IL-8 (5, 25, ng/ml) and specific polyclonal anti-il-8 antibody (10 g/ml, IL-8 Ab). Mesothelial and mesothelioma cell lines were immunostained also to show their IL-8 content in that way to. In vivo model Athymic nude mice (60) were intrapleural injected by human malignant pleural mesothelioma cells (CRL-2081). Animals were equally divided into three groups. The first group did not receive any treatment except tumor cells (tumor group). The second group was intraperitoneal administered by tumor cells and specific neutralizing anti human IL-8 Ab (IL-8 Ab) in every 48 hours (Ab group). The third group was given by tumor cells and aspecific mouse preimmune IgG as treatment in each 48 hours in a similar way (control Ab). Animals received intraperitoneal injection of IL-8 Ab, control Ab or no treatment respectively, every 48 hr for 15 days. Body weights of mice were monitoring continuously. 5,10, 15 days after injection 6-6 mice of each group were sacrificed. At the time of death peripheral blood was collected from Opthalmic artery. Tumors were dissected from the mice and weighted in mg. Complete autopsy was performed to examine the extent of tumor spread.

4 Pleural fluids and serum IL-8 levels (ELISA, pg/ml), tumor (mg) and body weight (gr) of mice were measured 5, 10, 15 days following tumor injection at sacrifice of 6 mice/group/time point. Groups Tumor cells injection Treatment Specific anti IL-8 Ab Aspecific IgG Ab 1. Tumor IL-8 Ab Control Ab Statistical Analysis All values are expressed as mean standard deviation. Data were compared using the Student s t-test and ANOVA. Values were considered to be statistically significant when p was less than 0.05 (p 0.05). Results Antigenic IL-8 levels in human pleural fluids Twelve patients with pleural effusions were evenly grouped (malignant mesothelioma and control: chronic heart failure) and studied. Six had pleural effusions secondary to mesothelioma (4 mixed, 1 epithelial, 1 sarcomatous). IL-8 levels were significantly (p ) elevated in patients with malignant mesothelioma ( ng/ml) compared with patients with CHF ( ng/ml). Detection of IL-8 in human mesothelioma and mesothelial cell culture supernatants All 3 mesothelioma cell lines, tested by ELISA, produced detectable levels of human IL-8 without stimulation. However, IL-8 was not detected in supernatants from mesothelial cells even after 96 hours of incubation in SFM. We observed significant, time dependent increases in IL-8 levels in all three mesothelioma supernatants. After 96 hours of incubation, IL-8 levels ranged between pg/ml in the CRL-5820 and pg/ml in the CRL-2081 cell line. Mesothelioma and mesothelial cell proliferation in response to IL-8 IL-8, in significant and dose-dependent manner, stimulated cell proliferation of CRL-2081 and 5915 mesothelioma cell lines as represented by [ 3 H]-thymidine incorporation. Maximal [ 3 H]-thymidine uptake occurred in response to 50 ng/ml of IL-8 (CRL-2081: % and CRL-5915: %, p 0.001, when compared with SFM, as control). The incubation with 100 ng/ml of IL-8 did not increase the [ 3 H]- thymidine incorporation above the level of 50 ng/ml IL-8 effect. The proliferation behavior of the CRL-5820 mesothelioma and the mesothelial (CRL-9444) cell lines, however, remained unaltered compared to untreated controls.

5 Furthermore, administration of specific polyclonal anti human IL-8 antibody (IL-8 Ab) demonstrated significant inhibition of the proliferation of CRL-2081 ( %, p 0.001) and CRL-5915 ( %, p 0.001) mesothelioma lines when compared with SFM. IL-8 (50 ng/ml) also stimulated and IL-8 Ab (10 g/ml) also inhibited the cell proliferation in CRL-2081 ( , p and , p 0.05) and 5915 ( , p 0.05 and , p 0.001) mesothelioma lines as demonstrated by direct (hemocytometer) cell count. There was no significant response to IL-8 as well as IL-8 Ab in the CRL-5820 and the mesothelial cell lines. Tumor model Throughout the study, 4 mice died due to the certain respiratory failure resulted from the advance tumor progression in growth and metastasis, but these occurred only in the non- and control Ab-treated (IgG) groups. In the IL-8 Ab-treated group, we found two animals without developed tumor mass at the day of sacrifice. Pleural and serum IL-8 levels in athymic nude mice IL-8 levels in both pleural fluids and serums increased significantly throughout the study. In non- and control Ab treated animals, the increase in IL-8 levels followed a similar pattern without significant difference between the two groups. The IL-8 levels were observed to be 6-10 fold elevated in pleural fluids when compared with serums during the 15 days. The IL-8 levels from IL-8 Ab-treated mice were significantly (p<0.0001) less than that in both non-or control Ab treated animals throughout the 15 days study period even in pleural fluids or serum. The effects of neutralizing IL-8 Ab on tumor growth The passive immunization with neutralizing IL-8 antibodies attenuated tumor growth. The tumor weights were 1.5 times elevated (p 0.007) in control Ab and untreated groups when compared with that in IL-8 Ab treated mice during the 15 days. Changes in body weights The weights of mice significantly decreased in all groups during the study period. While the baseline weights were similar among the animals, these became significantly lower (p 0.05) in either control Ab- or untreated mice when compared with Ab-treated group. Treatment 15 dys after tumor cells injection Specific anti IL-8 Ab Tumor cells injection Specific anti IL-8 Ab Body weight (g) 14,6±0,8 16,0±0,7 20,0±0,8 Tumor weight (mg) 507,9±37,9 524,6±24,8 342,4±22,9 Serum IL-8 (pg/ml) 603,2±17,2 612,7±22,1 43,7±6,5 Pleural IL-8 (ng/ml) 5,8±0,3 6,2±0.3 0,49±0,04

6 Correlation of tumor weight and pleural fluids IL-8 levels In a bivariate linear regression model using IL-8 level as predictor variable and tumor weight as the outcome variable, IL-8 was found to have a significant impact on tumor growth (p , r=0.88, r 2 =0.77). There was a significant and direct correlation between IL-8 levels and tumor weights of all animals enrolled in this study. Discussion Our results demonstrate that pleural fluids from patients with mesothelioma contained significantly greater IL-8 levels than did pleural fluid from CHF patients. As the source of these elevated levels of IL-8, we showed that all three mesothelioma cell lines constitutively expressed IL-8; however, there was no detectable IL-8 neither in the supernatants nor in the cells by immunostaining from resting mesothelial cells. It is interesting that the CRL-2081 mesothelioma cell line produced far the highest levels of IL-8 (ng/ml versus pg/ml) compared to other (CRL-5915 and CRL-5820) cell lines and became the most aggressive tumor regarding proliferation capacity and prognostic behavior in transplant model (data not shown). We also studied the effect of exogenous IL-8 and the requirement of IL-8 for growth and proliferation in mesothelioma and mesothelial cells as measured by the [ 3 H]-thymidine incorporation and direct cell counts. Treatment with exogenous human IL-8 showed a significant, dose-dependent increase in cell proliferation in CRL-2081 and CRL-5915 mesothelioma and anti-il-8 antibody was able to decrease in a significant fashion the proliferation of these cell lines. Neither [ 3 H]-thymidine incorporation nor cell counts changed after either addition of exogenous IL-8 or anti IL-8 antibody in the CRL-5820 mesothelioma and mesothelial cell lines where the IL-8 production was nearly undetectable or absent. These findings suggests that the lack of IL-8 binding sites on these cells may be responsible for their unresponsiveness to IL-8. Our results demonstrate, that treatment with neutralizing antibody against IL-8 protein effectively decreased tumor growth of human malignant pleural mesothelioma in athymic nude mice model when compared to control antibody- or untreated animals. In our work, we report a new mesothelioma animal model in which the injection of human CRL-2081 type malignant pleural mesothelioma cells into pleural cavity of nude mice cause tumor development in 96.7%. We described a more patient-like model compared to previous either asbestos induced intrapleural or subcutaneous injected mesothelioma models. The microenvironments of subcutaneous injected tumor cells or transplanted tissues are radically different than that in pleura, and as a result of that the biologic behavior and metastatic potential of developed tumor or response to treatments might be altered. In our model, the development of neoplasm is faster, takes higher rates and the tumor is human origin compared to the inhalation or intrapleural injected asbestos models. We also have to state that immundepleted recipient mice used in our model are still a modified microenvironments compared to human conditions, but more likely similar than others before. Our major and most important novelty might be that IL-8 antibody decreased both pleural and serum IL-8 levels as well as the tumor propagation as seen by the milder increase in tumor weight and milder decrease in body weight when compared the IL-8

7 Ab-treated groups to control Ab- or untreated animals. Moreover we found a strong, direct correlation between IL-8 levels of pleural fluids and the weight of tumor masses. In a study (not detailed in the thesis), we observed a very interesting finding, which could be connected to the data of our actual report. We showed that talcum, considered to be the most effective compound for pleurodesis, increased very much the programmed cell death (apoptosis), in vitro. The most aggressive cell line (CRL-2081), which produces the highest levels of IL-8 showed to be the most sensitive to talc treatment by increase of apoptosis. However the viability of mesothelial cells, which capacity for IL-8 release was not relevant, were almost not changed after talc exposition. The question has to be raised about the possible correlation of talc produced increase in apoptosis and its probable effect in decreasing the levels of IL-8. This idea, however, has to be confirmed in a later study. Our results suggest, that talc produced pleurodesis not only a palliative procedure but it could be recommendable and effective treatment in deceasing the viability of tumor cells in malignant mesothelioma. Conclusion In conclusion, I would like point out our new results and potential clinical impacts of the aimed experiments: a. By inoculation of human malignant mesothelioma cells into pleural space, we created a new and more patient like animal compared to the previous subcutaneous models. b. We verified the increased IL-8 production in malignant mesothelioma. c. We also showed that at least - in subgroups of malignant mesotheliomas, IL-8 is an important autogenous growth promoting factor. The expansion of mesothelioma and the levels of IL-8 in pleural fluids and serum were found to be related. d. The treatment by IL-8 Ab diminished the progression of human malignant mesothelioma. e. Our results might have showed the way toward a new potential therapeutic option of human malignant mesothelioma by decreasing its IL-8 release and pleural level, which would increase the life expectancy and life quality of these patients.

8 List of publications used for the thesis I. G. Galffy, KA. Mohammed, N. Nasreen, MJ. Ward, VB Antony: Inhibition of interleukin-8 reduces human malignant pleural mesothelioma propagation in nude mouse model. Oncology Research 1999;11: II. III. IV. G. Galffy, KA. Mohammed, PA. Dowling, N. Nasreen, MJ. Ward, VB. Antony: Interleukin-8, an autocrine growth factor for malignant mesothelioma. Cancer Research. 1999;59: Gálffy G., Mohammed KA., Dowling PA., Ward MJ., Antony VB: Interleukin-8, mint direkt növekedési faktor malignus mesotheliomában. Medicina Thoracalis, 1999;52: N. Nasreen, KA. Mohammed, PA. Dowling, MJ. Ward, G. Galffy, VB. Antony: Talc induces apoptosis in human malignant mesothelioma cells in vitro. American Journal of Respiratory and Critical Care Medicine 2000;161: Abstracts related to the thesis V. Gálffy G: Az interleukin-8 szerepe a malignus mesothelioma progressziójában, Fiatal Immunológusok II. Foruma, Budapest, Szept. 28. VI. VII. VIII. G.Galffy, Mohammed KA, Nasreen N, Antony VB, Lantos A, Magyar P: The effect of interleukin-8 in malignant mesothelioma propagation in vivo. Finno-Ugric Conference on Pulmonology, Parnu, Estonia, May , Gálffy G, Antony VB, Magyar P.: Az interleukin-8 ellenes antitest kezelés és a malignus mesothelioma progresszió kapcsolata nude egér modellben Magyar Tüdőgyógyász Társaság 51. Nagygyűlése Ápr , Budapest. G. Gálffy, KA. Mohammed, N. Nasreen, MJ. Ward, VB. Antony: Inhibition of interleukin-8 reduces human malignant pleural mesothelioma propagation in nude mouse model. The European Respiratory Journal. 1999;14: S. IX. G. Gálffy, KA. Mohammed, PA. Dowling, N.Nasreen, MJ. Ward, VB.Antony: The potential role of interleukin-8 in human malignant mesothelioma progression. Lung Cancer 1999;25:S25. X. Gálffy G: A malignus pleurális mesothelioma propagációjának gátlása IL-8 ellenes antitest kezeléssel nude egér modellben. (Tüdőgyógyászati Allergológiai és Immunológia Megbetegedések Konferenciája, TAIM, Aug. Debrecen. XI. XII. XIII. Gálffy G, Mohammed KA, Antony VB,: Interleukin-8, mint direct növekedési factor malignus mesotheliomában Magyar Tüdőgyógyász Társaság 50. Nagygyűlése, Balatonfüred, okt.1-3. Medicina Thoracalis, Suppl :S18. G. Gálffy, MJ. Ward, PA. Dowling, KA. Mohammed, N. Nasreen, VB. Antony: Interleukin-8, an autocrine growth factor for malignant mesothelioma. Am. J. Respiratory and Critical Care Medicine, 1998;157;A740. Nasreen, KA. Mohammed, MJ. Ward, PA. Dowling, G. Gálffy, VB. Antony: Talc induces apoptosis in human malignant mesothelioma cells in vitro. Am. J. Respiratory and Critical Care Medicine, 1998;157:A63.

9 Acknowledgement I am grateful to Professor Magyar Pál to let me start the fellowship program abroad, which enabled me to produce my research work and encouraged me finishing this dissertation. As a consultant, he helped me a lot in professional questions. I want to show my gratitude to dr. Margit Abonyi (Semmelweis University, I. Depatment of Internal Medicine) for announcing me to the Hungarian American Fellowship program, and for aiding in Hungary and in the United States by even the smallest matter. I want to express my appreciation to Professor Veena B. Antony, (VA Hospital, Indianapolis, USA) and my Colleges, Friends abroad, who showed me techniques of experimental research works and supported my own project by providing funds and supplying professional background. I am also grateful to Professor George Sarosi (Indianapolis, USA), who helped me to get contact to Prof. Antony and get a position in the Research Department of VA Hospital. He was the one, who has always been friendly and took off my soul from the ground and kept my enthusiasm accurate enough to go over and continue my work far away from home. Last but not least, I want to express my special thanks to my family and my children to endure these hard times, when I had just no time for them, to my husband, who has always been there when we needed and also helped a lot.