WWIWWW! i §§ {IWHiWWHhH} .THS . M315 Ifiiiliu'iiifiliiiifltiiifiiiifliififijifliil 3 1293 02058 62 This is to certify that the dissertation entitled IMMUNOSTIMULATION BY NOVEL PLATINUM ANTICANCER AGENTS presented by Heather Jean Muenchen has been accepted towards fulfillment of the requirements for Ph.D. , Zoology degree in swabsgfing’Vg‘ Major professor Date 06/04/99 MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE MRS 2 ‘3 iii" 11m chlRC/DaleDuopGS-p.“ IMMUNOSTIMULATION BY NOVEL PLATINUM ANTICANCER AGENTS By Heather Jean Muenchen A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY Department of Zoology l 999 ABSTRACT IL'IMUNOSTIMULATION BY NOVEL PLATINUM ANT ICAN CER AGENTS By Heather Jean Muenchen Cisplatin [cis-diamminedichloroplatinum (II); CDDP] is an antineoplastic dmg with demonstrated activity against mainly ovarian and testicular cancers. Although cisplatin is an effective chemotherapeutic drug, it has very severe toxic side effects, of which gastrointestinal and nephrotoxicity are the dose limiting factors. Poly-[(trarLs-IJ- diaminocyclohexane) platinum]-carboxyamylose (“poly-plat”), 5-sulfosalicylato-mns- (1,2-diaminocyclohexane) platinum (SSP), and 4-hydroxy-a-sulfonylphenylacetato (tms—lJ-diaminocyclohexane) platinum (11) (SAP) are analogs of cisplatin with higher efficacy and potency, while eliciting less toxicity. Normal murine peritoneal macrophages, when treated with “poly-plat” and SSP (5 ug/ml) for 2 h at 37°C in culture, developed extension formations within 2 h. These cytoplasmic extensions radiate from the cell body in all directions. While SAP (5 ug/ml) treated macrophages, not showing any extension formation, instead assumed a discoid shape similar to that of the normal macrophages. Cisplatin (5 [Lg/ml) treated macrophages also showed similar extension formation but only after 24 h of treatment. When these drug treated macrophages are co-incubated with 8180 tumor cells they immediately establish contact with several target cells and form cytoplasmic continuity through which lysosomes are transported into the tumor cells causing their lysis. CDDP treated cells also form cytoplasmic extensions that are fewer in number and establish contact with fewer tumor cells when compared to “poly-plat” treated macrophages. Normal macrophages do not form cytoplasmic extensions and, when co- incubated with 8180 cells, fail to show any interaction. Based on fluorescence measurements after acridine orange labeling, we observed a 500-fold increase in the number of lysosomes in the macrophages after only 2 h of “poly-plat” treatment compared to normal cells. SSP and SAP both demonstrated a 100— fold increase in the number of lysosomes. Comparatively, cisplatin treatment demonstrated only a 50-fold increase in the lysosomes. Significant increases in TNF-a levels were observed in the supematants from SSP (5 ug/ml) treated macrophages at all times tested, with the most significant increase occurring at 2 h post-treatment (3250 pg/ml). “Poly-plat” and SAP (5 ug/ml) demonstrated very little TNF-a activity, barely reaching 200 pg/ml at 24 h post-treatment. Cisplatin demonstrated the usual enhanced release of TNF—a at various time intervals, reaching a peak value at 2 h post-treatment (3000 pg/ml). Compared to cisplatin treatments there was an increase in IL-la levels in the supematants of macrophages treated with either “poly-plat”, SSP, or SAP (5 ug/ml) for up to 24 h of testing. Dedicated to my son J adan. iv ACKNOWLEDGMENTS I wish to express my deepest thanks to Dr. Surinder K. Aggarwal, my advisor and mentor, from whom I have learned much in the areas of science and non science. His guidance, support, and patience has been greatly appreciated. I also wish to thank Drs. Will Kopachik, Thomas Corner, and Ashir Kumar for having served as my committee members. Lastly, I would like to acknowledge my family and friends whose support has enabled me to reach my goals. TABLE OF CONTENTS LIST OF FIGURES .................................................................... vii CHAPTER 1: INTRODUCTION ................................................... 1 CHAPTER 2: Activation of murine peritoneal macrophages after..............5 cisplatin and taxol combination CHAPTER 3: Enhanced immunostimulation by novel platinum .............. 12 anticancer agents CHAPTER 4: Immune system activation by cisplatin and its analog. . .......l9 “poly-plat”: an in vitro and in vivo study CHAPTER 5: Enhanced immune system activation after treatment .......... 27 with novel antineoplastic platinum agents CHAPTER 6: CONCLUSIONS .................................................... 34 vi LIST OF FIGURES CHAPTER 2 FIGURE 1 .............................................................................. 8 FIGURE 2 .............................................................................. 9 FIGURE 3 .............................................................................. 9 FIGURE 4 .............................................................................. 9 FIGURE 5 ............................................................................ 10 CHAPTER 3 FIGURE 1 ............................................................................ 14 FIGURE 2 ............................................................................ 15 FIGURE 3 ............................................................................ 16 FIGURE 4 ............................................................................ 17 FIGURE 5 ............................................................................ 17 CHAPTER 4 FIGURE 1 ............................................................................. 22 FIGURE 2 ............................................................................. 22 FIGURE 3 ............................................................................. 23 FIGURE4 ............................................................................. 23 FIGURE 5 ............................................................................. 24 FIGURE 6 ............................................................................. 24 FIGURE 7 ............................................................................. 24 vii CHAPTERS FIGURE 1 ............................................................................. 29 FIGURE 2 ............................................................................. 30 FIGURE 3 ............................................................................. 30 FIGURE 4 ............................................................................. 31 FIGURE 5 ............................................................................ 31 FIGURE 6 ............................................................................ 31 viii CHAPTER 1: INTRODUCTION INTRODUCTION Cisplatin [cis-diamminedichloroplatinum (II); CDDP], a broad spectrum anticancer drug, is proven to be effective in the treatment of testicular, ovarian, prostate, bladder, head and neck, and lung cancers.1 DNA denaturation is one of the accepted methods of its mechanism of action through its intrastrand and interstrand cross-links interfering with DNA replication and transcription. Another mechanism of action is through the activation of the immune system.2 The activation process includes the generation of extracellular products including interleukin-la (IL-la) and tumor necrosis factor-oz (T NF-a).3 CDDP has been shown to activate murine peritoneal macrophages in vitro.4 These activated macrophages seek out tumor cells through the formation of cytoplasmic extensions and lysosomal transfer to these target cells causing cell death.5 Although cisplatin is an effective anticancer drug, it has very severe toxic side effects, of which gastrointestinal and nephrotoxicity are the dose limiting factors.6 Taxol has a unique mechanism of antitumor activity in that it binds to a protein, tubulin, thus inhibiting cell division:7 CDDP plus taxol in combination has become increasingly prevalent in clinical treatment.7 Combination therapy is capable of producing very high response rates and is seemingly less toxic than either of the two drugs when administered alone.7 Although, the mechanism of action for the cytotoxic interactions between CDDP and taxol have not been determined. Poly-[(nans-1,2-diaminocyclohexane) platinuml-carboxyamylose (“poly-plat”), S-sulfosalicylato-trans-(1,2-diaminocyclohexane) platinum (SSP), and 4-hydroxy-0t- sulfonylphenylacctato (trans-LZ-diaminocyclohexane) platinum (11) (SAP) are second generation analogs of cisplatin with higher efficacy and potency. while eliciting less toxicity.8 This is particularly true of “poly-plat” which contains 1/5 the platinum of cisplatin.8 The possibility of drugs enhancing the immune system with less toxicity is very encouraging. “Poly-plat” is curative in a range of solid tumors including renal, breast, ovarian, plasma cell mycloma, and adenocarcinoma.8 It has been shown to be up to 15X as active as equimolar amounts of cisplatin, showing inhibition in many tumors where cisplatin is ineffective. 8 SSP is curative of many cisplatin resistant tumors including M5 ovarian and several plasma cell myclomas. SSP also elicits no nephrotoxicity and is less toxic than cisplatin even at LDlo dose (180 mg/kg).8 SAP, the most potent of the platinums, exhibits a wide spectrum of anti-tumor activity. It has facile reproducible synthesis and minimum acute toxicity at any effective dose from 1.85 mg to 15.0 mg/kg. We have here explored the effects of cisplatin plus taxol, “poly-plat”, SSP, and SAP on the macrophages so as to understand their mechanism/s of action in enhancing the immune system. REFERENCES 1. Nicolini M. Platinum and other metal coordination compounds in cancer chemotherapy. Boston: Martinus Nijhoff; 1990. 2. Roberts J, Pascoe J. Cross-linking of complementary strands of DNA in mammalian cells by antitumor platinum compounds. Nature 1972;235:282. 3. Palma J, Aggarwal S. Cisplatin and carboplatin mediated activation of murine peritoneal macrophages in vitro: production of Interleukin-1 alpha and tumor necrosis factor~alpha. Anti-Cancer Drugs 1995;6:1. 4. Singh S, Sodhi A. Interaction between cisplatin treated macrophages and Dalton's lymphoma cells in vitro. Exp Cell Biol 1989;56:1. 5. Palma J, Aggarwal S, Jiwa A. Murine macrophage activation by cisplatin and carboplatin. Anticancer Drugs 1992;3z665. 6. Walker E, Gale G. Methods of reduction of cisplatin nephrotoxicity. Ann Clin Lab Sci 1981;11:397. 7. Rowinsky E, Citarrdi M, Noe C, Donehower R. Sequence-dependent cytotoxic effects due to combinations of cisplatin and the microtubule agents taxol and vincristine. J Cancer Res Clin Oncol 1993;119:727. 8. Fiebi g H, Dress M, Ruhnau T, Misra H, Andrulis P, Hendrik H. GB-21, a novel platinum complex with antitumor activity in human renal and mammary xenografts. Proc Am Ass Cancer Res 1996;37:297. CHAPTER 2: ACTIVATION OF MURINE PERITONEAL MACROPHAGES AFTER CISPLATIN AND TAXOL COMBINATION Anti-Cancer Drugs 199"; 8. pp. 784-789 Activation of murine peritoneal macrophages after cisplatin and taxol combination Heather J Muenchen and Surinder K Aggarwal Department oi Zoology. Michigan State University. East Lansing. MI 48824-1115. USA. Tel: (+1) 517 353-2253; Fax: (+1) 517 432-2789. Cisplatin and paclitaxel are potent antineoplastic agents. Their distinctly ditterent mechanisms of action have prompted laboratory and clinical research into their use in combination therapies. Murine peritoneal macrophages heated with cisplatin and paciitaxel in combination elicit an increase in their number of lysosomes. Drug—treated macro- phages, when co-incubated with sarcoma 180 cells. establish cytoplasmic contact and transfer lysosomes into tumor cells earning tumor cell lysis. in addition, analysis of tissue cuitura supematants show increased levels of interleukin-1: and tumor necrosis factor-z. Our study shows that cisplatin and paclitaxel in combination enhance elements of the immunesystamwithgreataramcacyandpotencythanwhen used alone. Key words: Cisplatin, interleukin~1z. lysosomes. in vitro. macrophages. taxol. tumor necrosis factor-a. Introduction The remarkable clinical efficacy of paclitaxel (taxol) has resulted in numerous observations of partial and complete remission of advanced ovarian cancer in women.1 Recently, reports of the efficacy of the drug in breast. lung and prostate cancer have aroused great interest in the antitumor compound.‘ Taxol has a unique mechanism of antitumor activity in that it binds to a protein. tubulin. thus inhibiting cell division.‘2 Cisplatin [cis-diamminedichloroplatinum (II); CDDP]. a heavy metal platinum coordination complex. is proven to be effective in the treatment of testicular. ovarian. prosrate, bladder. had and neck. and lung cancers.’ DNA denamration is one of the accepted methods of its mechanism of acrion through its intrastrand and interstrand cross-links interfering with DNA replication and transcription.“ Another mechan- ism of acrion is through the activation of the immune system. Acrivated macrophages have been found to effectively destroy tumor cells by cytotoxic mechan- isms."6 The acrivation process includes the generation of extracellular products including interleukin (HM: and tumor necrosis factor (IND-31.- Correspondence to SK Aggarwal 784 Ami-Cancer Drugs - Vol 8- 199* Cisplatin plus taxol in combination has become increasingly prevalent in clinical treatment.8 Combi- nation therapy is capable of producing very high response rates and is seemingly less toxic than either of the two drugs when administered alone.8 Although. the mechanism of aetion for the cytotoxic interacrions between cisplatin and taxol have not been determined. We have here explored the effects of cisplatin plus taxol on macrophages so as to understand their mechanism of action in the immune system enhancement and its efficacy in fighting cancer. Materials and methods Cell cultures Swiss Webster mice (Charles River. Location. MA) were sacrificed by cervical dislocation and peritoneal macrophages were isolated by injecrion of 5 ml chilled minimal essential medium (MEM: Gibco. Grand Island. NY) without serum containing 1% antibiotic-antimy- cotic [penicillin G (10 000 U/ml). streptomycin sulfate (10 000 rig/ml) and amphotericin B (25 pg/ml); Gib- co] into the peritoneal cavity. After gently massaging the abdominal wall. cells were aspirated and seeded onto 18 mm2 glass coverslips. placed in 35 mm Petri dishes. at 2-4 x 10“ cells/ml and incubated for 2 h at 37C. Coverslips were washed vigorously to remove non-adherent cells. Cell cultures were incubated in normal medium (minimal essential media and 10% heat-inactivated fetal calf serum) at 373C in a 5% C02 incubator. Sarcoma 180 ascites ($180: CCRFS-IBOII; American Type Culture Collection. Rockville. MD) were maintained in culture using normal medium. Cells were washed with Hank's balanced salt solution (HBSS: Gibco) and centrifuged at 1000 g for 5 min for use in experiments. These cells served as target cells for macrophages and were added to cultures at 3 x l0S cells/ml concentration. I [99‘ Rapid .Screna' Publishers Treatments Cisplatin and taxol were prepared in 0.85% NaCl in 5 and 10 rig/ml concentrations, respectively. Macro phages were treated with the drugs for 2 h. The drug(s)-containing medium was replaced by normal medium. and supernatant (500 ill) was collected at 0.5, 1. 2 and 24 h for cytolytic facror/s analysis. Untreated cells in normal medium served as controls. lL-11 assay 11:11 was assayed using an ELISA kit (Genzyme. Cambridge. MA). The method used the multiple anti- body sandwich pn'nciple,9 where monoclonal anti- mun'ne IL-lat was used to bind mun'ne IL-lor present in the supernatant. A biotinylated polyclonal antibody binding the [blot was added and unbound material was washed out. Peroxidase-conjugated avidin was used to bind these biotin tagged complexes. A substrate solution was then added resulting in a color change. The reaction was stopped by acidification and absor- bance was read at 450 nm. Standard curves were generated with 11:11 ( 15-405 pg/ml) provided in the kits and linear regression analysis was performed. TNF-ar assay TNF—ar released from supematants of the macro- phages was assayed using a specific analysis kit (Genzyme). Again. the multiple antibody sandwich principle9 was utilized with a murine monoclonal antibody specific for murine TNF-ar in the samples. A horseradish peroxidase-conjugated anti-murine TNF-z antibody was used to bind the multiple epitopes on TNF-ar. A subStrate solution was then added resulting in a color change. The reaction was Stopped by acidification and absorbance was read at 450 nm. Standard curves were generated with TNT-a (35- 2240 pg/ml) provided in the kits and linear regres sion analysis was performed. Lysosomal assay The quantitation of lysosomes before and after various treatments was achieved by exposing macrophage cultures to fresh medium containing acridine orange (5 rig/ml) for 30 min at 37‘C in the dark.” After careful washing. macrophages were examined under a Zeiss 10 laser scanning confocal microscope and visual counts made. Cisplatin. taxol and macrophages Macrophage-tumor cell interaction studies To study macrophage-tumor cell interaction. macro- phage monolayers were treated with either cisplatin (5 rig/ml). taxol ('10 rig/ml) or cisplatin (5 pg/ml) plus taxol (10 rig/ml) for 2 h at 35C in a 5% C03 incubator. The medium was then replaced by normal medium and the 5180 tumor cells were added. Macrophages and tumor cells were co-incubated for 2 h. Coverslips seeded with macrophages and tumor cells were fixed with 1.5% glutamldchyde on 0.05 M phosphate buffer (pH 7.2) at room temperature for 10 min. Macrophage-tumor cell interaction was viewed using phase contrast microscopy. Resuns lL-l a: release Compared to normal there was a gradual increase in IL- 11 levels in the supematants of macrophages treated with cisplatin (5 rig/ml) plus taxol (10 rig/ml) (Figure 1). There was a gradual increase in macrophages treated with cisplatin alone. However. taxol-treated macrophages demonstrated a large increase (400 pg/ ml) in 11-13: after 30 min post-treatment. These levels gradually decreased until 2 h post-treatment. After 24 h again there was an increase reaching approxi- mately -75 pg/ml. TNF-at release Compared to normal. a combination treatment of the two drugs cisplatin (5 rig/ml) plus taxol (10 rig/ml) demonstrated increased levels of TNT-a after only 30 min posr-treatment. A cyclical release was observed when TNF-at was viewed at l. 2 and 24 h post- treatment. This cyclical release pattern was also true for both cisplatin and taxol when used alone. but the levels of TNF-ar were not as high as after the combination treatment (Figure 2). Macrophage activation Murine peritoneal macrophages demonstrated exten- sion formations after 2 h post-treatment with cisplatin alone (5 pg/ml) (Figure 3A). However. taxol (ll) rig/ml) and cisplatin (5 ,ug/ml’) plus taxol (10 [lg/ml) treated macrophages did not show any extension formation. for up to 24' h. but instead assumed a discoid shape similar to that of the normal macrophages (Figure 38-D). AnnCancer Drugs - Vol 8 - [99' 785 H] Muenchen and 5K Aggarwal Lysosomal studies Based on fluorescence measurements after acridine orange labeling, a loofold increase in the number of lysosomes in the macrophages was observed only after 2 h of cisplatin (5 rig/ml) plus taxol (10 rig/ml) treatment (Figure 4A) as compared to untreated cells (Figure 48). Both cisplatin and taxol alone demon- strated only a 50-fold increase in the number of lysosomes (Figure 4C and D). Macrophage-tumor cell interaction studies Macrophages cocultured with tumor cells ($180) establish cell-cell contact within 30 min and demon- strated a transfer of their lysosomes into the tumor cells through moplasmic continuity assumed after co- incubation. Cisplatin-treated macrophages have been shown to transfer their lysosomes down the cytoplas- mic extensions (Figure 5A). As stated earlier taxol and cisplatin plus taxol treated macrophages assume a discoid shape without cytoplasmic extensions. This. 400 T 350 T —-T 300 "l’ 250 " 200 «. lL-1a Concentration (pg/ml) 100 ‘- 50i- 0.5 1 however. did n0t efi‘ect the ability of macrophages to establish contacr with the tumor cells and transfer their lysosomes (Figure SB and C). These tumor cells eventually undergo lysis. Untreated macrophages never established contact with tumor cells (Figure SD). Discussion The antineoplastic activity that taxol has demonstrated in advanced ovarian cancer and other neoplasms in which the platinum analogs are among the most active agents has been the impetus for the deveIOpment of taxol plus cisplatin combination regimens.8 Taxol and cisplatin are the two most effective agents discovered to date for treating advanced-stage cancers. “ Learning how best to combine these agents is the focus of preclinical and clinical studies conduCted at a number of institutions. The overt effects of the anticancer drugs cisplatin and taxol appear to be DNA modifica- tion and microtubule stabilization. respectively. yet the mechanisms by which these drugs elicit tumor cell I Control I Cisplatin El Taxol B Cisplatin + Taxol WWWM 2 24 Various drug post-treatment (h) Figurai. Bargraphshowing IL-1zreleaseinthesupernatantsoimrineperitonaal macrophagestreatediorzriwimeither cisplatin (5 ug/ml).taxol(10pglml)orcisplatin piustaxoialterao min. 1.2and24hpost—treatment.Notethelarge increases! wmmmmmmd.BomaspiafinaMcispiafiriplustaxdshowagaanlmBasemmeevelsdIL-ta.reaching amazinmievelatZlihpost—treatmant 786 Ami-Cancer Drug: . Vol 8- 199? 3500 T F? jg; 3000 4 . 7...; B .. 1.. 2 2500 -- E . F» f: 3 :_ ‘ 3 2000 - ~55: E —: E 5.. o ,—.._. g ; o 1500 -- g) , U its. ’- 1000 a ,5; ' i; 3 . Cisplatin. taxol and macrophages I Control I Cisplatin Cl Taxol El Cisplatin . Taxol k:- 5:124 Ti 0.5 1 2 24 Various drug post-treatment (h) Figure 2. Bar graph showing TNF-a release in the supernatant of murine peritoneal gestreatediorZhwitheither cisplatin (5 rig/ml) taxol (10 rig/ml) or cisplatin plus taxol after 30 min 1 2 and 24 h post- -treatment Note the maximum increase in TNF-a Figure 3. Light micrographs showing macrophages hot treatment with cisplatin (A) taxol (B). cisplatin plus taxol (C) and normal (D).Note extension ionnationait cisplatin treatment (arrows). Taxol and cisplatin plus taxol treated cells show mostly a discoid shape similar to that of normal macrophages. Bar=0.5 mm. death are not well understood.” Both in nine and in vivo studies conclude that taxol interacts synergisti~ eallv with cisplatin in a manner that is highly schedule dependent. torcisplatin plus taxol at 2 h post-.treatment The release of TNF-z appears to be cyclical in all drug treatmentswithcisplatinplustaxolshowingthehighestaetivity. C Figure4. Fluorescentimagestakeniromtheleisstmaser scanning confocal rnicrowope of macrophages labeled with acridine orange (5 uglml) showing noe in cisplatin plus taxol (A) normal (8")9 cisplatin (C) and taxol (0). Note the increase in fluorescence alter cisplatin plus taxol treatment (arrows). Bar: 10 um. Arm—Cancer Drug: . rm 8. 199‘ 787 H] Muenchen and SK Aggarwal Figure 5. Phase contrast images taken from the Zeiss 10 laser scanning confocal microscope of macrophages co-incubated with $180 tumor cells (arrowheads) after 2 h of treatment with cisplatin (A). taxol (B). cisplatin plus taxol (C) and normal (0). Note that taxol and cisplatin plus taxol treated macrophages do not show long cytoplasmic extensrons so very characteristic of cisplatin. However, treated cells are still able to establish contact with tumor cells and destroy them through lysosomal transfer (arrows). Bar=10 Mm. Cisplatin stimulates immune responses by activating monocyte-macrophages and other cells of the immune system.” We have demonstrated here three mechan~ isms by which the immune system is enhanced by cisplatin and taxol combination compared to cisplatin or taxol treatments: (i) release of cytolytic factors [1:101 and TNF-at. (ii) increase in the macrophage lysosomes, and (iii) cell—cell recognition through contact between activated macrophages and tumor ceUs. lL-la was first described as a lymphocyte activating factor because of its ability to stimulate T cells." lts release by activated macrophages and its cytotoxicity to tumor cells suggests lL-la as a potent mediator in 788 Arm-Cancer Drug: - Vol 8 - I997 tumor ceu killing by macrophages.” Our study demonstrates taxol as having the greatest increase in IL-lat after 30 min. while decreasing by 24 h. but yet stays higher than the untreated macrophages. Both cisplatin and cisplatin plus taxol in combination showed a gradual increase in lL-la reaching a maximum level after 24 h post-treatment increased levels of TNF—a have also been observed in activated macrophages. TNF-or is known to mediate a variety of functions which include host defense mechanisms and growth, H Recent studies have shown increased levels of TNF-a after cisplatin treatment. It is apparent that TNFJJ is another important mediator of tumor cell killing by macrophages. Cisplatin plus taxol combination demonstrated the greateSt increase in TNF- a. reaching a maximum level after only 30 min. All three treatments seemed to cause a cyclical release of TNF-a. Tumor cell death through macrophage aetivation includes the production of lysosomes'm‘ Previous studies have shown cisplatin-treated macrophages to increase their number of lysosomes and transfer them via cytoplasmic extensions into tumor cells.” This transfer results in eventual lysis of tumor cells.19 Cisplatin plus taxol combination showed the greateSt increase in the level of cytoplasmic lysosomes with a lOO-fold increase over that of untreated macrophages. Both cisplatin and taxol independently showed approximately a 50-fold increase in lysosomes. Lysosomes are only released when cytoplasmic continuity between the macrophage and tumor cell has been eStablished. Past studies have shown that cisplatin aetivates macrophages to form cytoplasmic extensions which make contact with tumor cells.”J Through these extensions lysosomes are transferred and cell death occurs. Although taxol and cisplatin plus taxol combination did n0t aCtivate macrophages to form cytoplasmic extensions they still established contact and formed a cytoplasmic continuity with the tumor cells. Through this cytoplasmic continuity there was an apparent transfer of lysosomes from macro- phage to tumor cell and eventual cell lysis. Conclusion Cisplatin plus taxol combination stimulates various cytolytic factors of the immune system better than when these drugs are used independently. The macrophage activation includes production of cyto- plasmic lysosomes. macrophage-tumor cell contact. and release of cytolytic factors IL-1 1 and TNF-z. Past in vitro and in vivo Studies have shown that when these two drugs are used in combination they are more effective and less toxic than when they are used separately. Our results support activation of the immune system as an additional mechanism of action of this combination therapy. We also propose. based on our observations, that cisplatin plus taxol combina- tion activates various cytolytic factors of the immune system better than Cisplatin or taxol independently. References 1. Wall ME. Wani MC. Camptothecin and taxol: discovery to clinical—the thirteenth Bruce F Cain Memonal Award 11 Cisplatin. taxol and macrophages Lecture. Cancer Res 1995; 55: ‘55. . Haiek R. Paclitaxel Cas-Lek-Cesk 1996. 135: 393 3. Nicolini M. Platinum and other meta! coordination compounds in cancer cbemotberam'. Boston: Martinus Nijhofl‘ 1990. -i. Roberts J). Pascoe JM. Crossrlinking of complementary strands of DNA in mammalian cells by antitumor platinum compounds. Nature 19'2: 235: 282. 5. Adams D. Hamilton T. Acrivation of macrophages for tumor ceU kill: Effector mechanisms and regulation. in: Heppner GH. Fulton AM. eds. .llampbages and cancer Boca Raton. FL: CRC Press 1988: 2‘ (i. Carleton S. Stevenson A. Hibbsj. EffeCtor mechanisms for macrophage-induced cytosmsis and Cytolysis of tumor cells. in: Heppner GH. Fulton AM. eds. .llacropbages and cancer. Boca Raton. FL‘ CRC Press 1988; 39. ". Palam JP. Aggarwal SK. Cisplatin and carboplatin mediated activation of murine peritoneal macrophages in vitro: producuon of interleukinl and tumor necrosrs factor. Anti-Cancer Drugs 1995; 6: 1. 8. Rowinsky ER. Citarrdi M). Noe CA Donehower RC. Sequence-dependent cytotoxic effects due to combina- tions of cisplatin and the antimicrotuble agents taxol and vincristine. J Cancer Res CIr'n Oncol 1995; 119: '2‘. 9. Meager A. RlA. IRMA. and ELISA assays for cytokinse. in: Ballm'ill F. ed. Ci'tolzines: a practical approach. Oxford: Oxford University Press 1991: 299. 10. Poole A. The detection of lysosomes by vital staining with acridine orange. In: Dingle). ed. L_i‘sosomes. a laboratory bandboole. Amsterdam: Elsevier/North Holland Biomedi- cal Press 19": 513. 11. Parker R1. Dabholltar MD. bee KB. Bostick-Bruton F. Reed E. Taxol effeet on cisplatin sensitivity and cisplatin cellular accumulation in human ovarian cancer cells. .llonogr Natl Cancer Inst 1993; 15: 85. ll. Donaldson KL Goolsby GL Wahl AF. Cytotoxicity of the anticancer agents cisplatin and taxol during cell prolifera- tion and the cell cycle. Int] Cancer 1994: 57: 84". 15. Kumar R Shn'vaStava A. Sodhi A Cisplatin Stimulates protein tyrosine phosphorylation in macrophages. BIO- chem Mol Biol Int 1995: 35: 541. 14. Gen 1. Waltsman B. Potentiation of cultured mouse thymocyte response by factors released by peripheral leukocytes. j Immunol 1986: 107: 1""8. 15. Palma .lP. Aggarwal SK. Cisplatin and carboplatin mediated release of otolytic famous in murine peritoneal macrophages in vitro. Anti-Cancer Drugs 1994: 5: 615. 16. Bucana C. Hover L Hobbs B. et a1. Morphological evidence for translocation of lysosomal organelles from cy'tOtoxic macrophages into the cytoplasm of minor target cells. Cancer Res 19‘6; 36: 444-1. 1". Hibbs J. Heteroqtolysis by macrophages activated by bacmus calmette guerin: lysosome exoqmsis into tumor cells. Science 19"4': 148: 468. 18. Palma JP. Aggarwal SK Jiwa A. Murine macrophage activation after cisplatin or carboplatin treatment. Anti- Cancer Drugs 1992; 3: (16%. 19. Singh S. Sodhi A. Interaction between cisplatin treated macrophages and Dalton‘s lymphoma cells m it'tm. Exp Cell Bu)! 1989; 56: 1. 14 (Received [0 july I99": accepted I”jul_r I 99") Ann-Cancer Drugs . 1'01 8 - 199' 789 CHAPTER 3: ENHANCED IMMUNOSTIMULATION BY NOVEL PLATINUM ANTICANCER AGENTS 12 Anti-Cancer Drugs 199‘. 8. pp. 525—328 Enhanced immunostimulation by novel platinum anticancer agents Heather J Muenchen, Surinder K Aggarwal, Hemant K Misra1 and Peter J Andrulis1 Department of Zoology. Michigan State University. East Lansing, MI 48824-1115. USA. Tel: (+1) 517 353-2253; Fax: (+1) 517 432-2789. ‘Andrulis Pharmaceuticals Corp. Beltsville. MD 20705, USA. ‘Pon-plat’, SSP and SAP are second generation analogs of cisplatin which have been shown to activate murine peritoneal macrophages in vivo and in vitro. Murine perito- heal macrophages treated with ‘pon-plat'. SSP or SAP (5 rag/mg) tor 2 h are stimulated to form cytoplasmic exten- sions. Drug-treated macrophages also elicit an increase in the number of lysosomes. In addition, analysis of tissue culture supematants shows increased levels of interleukin- 1a and tumor necrosis factor-a. These results show that ‘poly-plat', SSP and SAP enhance the immune system with greater efficacy and potency than cisplatin. Key words: Cisplatin. ‘poly-plat‘, interleukin-1a. in vitro. lysosomes. macrophages. SAP, SSP. tumor necrosis factor-a. Introduction Cisplatin [cis-diamminediehloroplatinum (II): CDDP]. a broad spectrum anticancer drug.I is proven to be effective in the treatment of testicular. ovarian. prostate. bladder. head and neck. and lung cancers.2 Activated macrophages have been found to effec- tively destroy target cells by cytotoxic mechan- isms}H The activation process includes the generation of extracellular products including inter- leukin (ID-Ia and tumor necrosis faCIor (TNF)-a.’ Although cisplatin is an effective anticancer drug. it has very severe toxic side effects. of which gastro- intestinal and nephrotoxicity are the dose-limiting factors.6 Poly-[( trans-l .2-diaminocyclohexane‘) platinum]- carboxyamylose (poly-plat"). S-sulfosalicylato-trans— (1.2-diaminocyclohexane) platinum (SSP) and 4-hy- droxy-a-sulfonylphenylacetato (trans-1 .2-diaminocy- clohexane) platinum (II) (SAP) are second generation analogs of cisplatin with higher efficacy and potency. while eliciting less toxicity. This is particularly true of ‘poly-plat' which contains one- fifth the platinum of cisplatin. The possibility of drugs enhancing the immune system with less Correspondence to SK Aggarwal (j [99' Rapid Science Publishers l3 toxicity is very encouraging. We have here explored the effects of ‘poly-plat‘. SSP and SAP on the macmphages so as to understand their mechanism(s) of action in enhancing the immune sy'Stem. Materials and methods Cell cultures Swiss webster mice (Charles River. Portage. M1) were sacrificed by cervical dislocation and perito- neal macrophages were isolated by injection of 5 ml chilled minimal essential medium (MEM; Gibco. Grand Island. NY) without serum containing 1% antibiotic—antimycotic [penicillin G (10000 U/ml). streptomycin sulfate (lOOOOag/ml) and ampho— tericin B (25 ‘ug/ml)] into the peritoneal cavity. After gently massaging the abdominal wall. cells were aspirated and seeded onto 18 mm2 glass coverslips. placed in 35 mm Petri dishes at 2—4 X 10°cellsfml and incubated for 2 h at 57°C after which cells were washed vigorously to remove non-adherent cells. Cell cultures were incubated in normal medium (minimal essential media and 10% heat-inactivated fetal calf serum) at 37°C in a 5% CO; incubator. Treatments ‘Poly-plat' was prepared in 0.85% NaCl while SSP and SAP (Figure 1) were dissolved in 0.85% NaCl and 0.1% NaC05 in 5 ,ug/ ml concentrations. Macro phages were treated with the drugs for 2 h. The drug(s)-containing medium was replaced by normal medium and supernatant (500 al) was collected at 0.5. 1. 2 and 24 h for cytolytic faCtor(s) analysis. In addition. macrophages were also treated with cispla- tin [S ‘ug/‘ml dissolved in physiological saline with 3 .ul/ml of dimethylsulfoxide (DMSO: Sigma. St Louis. MO)]. Untreated cells in normal medium served as controls. Anti-(Lancer Drugs - iii/s- 199' 323 H] Muenchen et al. Cisplatin Cis-diamminedichloroplatinum(ll) ‘Poly-plal' CH20H CHZOH I HC —0 CH—O / \ -0- HC (ISH-O— lCH CH—O" COOH COO COO COOH pt HzN NH2 Poly-[( trans-1 .2-diaminocyclohexane)platinum]-carboxyamylose 35p qwz/ NHZ \ Pt< 02C 0U SO3H 5-sullosalieylato— trans-(1 .2-diaminocyclohexane)platinum SAP 0 l —S Cc:::>m .<: _§>c~—@o~ II 0 4-hydroxy-a-sullonyiphenylacetato( trans 1 .2-diaminocyclohexane)platmum(lI ) Figure 1. Structure of compounds studied. IL-1a assay [Ltd was assayed using ELISA kits (Genzy me. Cam- bridge. MA) The method used the multiple antibody sandwich principle."J where monoclonal antiomurine [bid was used to bind murine lL—la present in the supernatant. A biotinylated polyclonal antibody bind 324 Anti-Cancer Drugs - Vol 8 . [99‘ 14 ing the lL—la was added and unbound material was washed out. Peroxidase-conjugated avidin was used to bind these biotin tagged complexes. A substrate solution was then added resulting in a color change. The reaction was Stopped by acidification and absorbance was read at 450 nm. Standard curves were generated with IL-la ( 15—405 pgg'ml) provided in the kits and linear regression analysis was per- formedg TNF-a assay TNF-a released from supernatants of the macro- phages was assayed using specific analysis kits (Genzyme). Again. the multiple antibody sandwich principle was utilized with a murine monoclonal antibody specific for murine TNF-a in the samples.8 A horseradish peroxidase-conjugated anti-mun‘ne TNF-a antibody was used to bind the multiple epitopes on TNF-a. A subStrate solution was then added resulting in a color change. The reaction was Stopped by acidification and absorbance was read at 450 nm. Standard curves were generated with TNF-a (35—22-10 pg/ml) pron‘ded in the kits and linear regression analysis was performedw Lysosomal assay The quantitation of lysosomes before and after various treatments was achieved by exposing macro phage cultures to fresh medium containing acridine orange (5 .ug/ml) for 50 min at 5"°c in the dark.” After careful washing macrophages were examined under a Zeiss IO laser scanning confocal microscope. Results IL-1a release Compared to cisplatin treatments there was an increase in IL—Ia levels in the supematants of macrophages treated with either ‘poly-plat‘. SSP or SAP (5 _ug/ml) for up to 24 h of teSting (Figure 2). The greatest increases were seen 2 h post-treament (400—500 pg/ml) with a subsequent decrease from there on. IL-la levels demonStrated a consistent increase after cisplatin treament. reaching a maxi- mum after 24 b. Although. lL-la levels after ‘poly- plat'. SSP or SAP treament demonstrated a decline after a peak at 2 h. these levels were still equal to or above those after cisplatin treatment. Platinum analogs and immune system 700 T 600 4- 500 ~- I Normal .............. g I Cisplatin 5 «)0 .0. U Poly-plat _5 I ssra 7% I SAP S 300 -r ;fij:j:j:;f; 5 ............. 200 s- ............... 100 "" ....... 0 - ....... 0.5 1 Various drugs post-treatment (h) Figure 2. Bar graph showing IL-1a release in the supematants of murine peritoneal macrophages treated with either cisplatin, ‘poly-plat'. SSP. or SAP (5 rig/ml) after 30 min. and 1. 2 and 24 h post-treatment. Note the large increase at 2 h post-treatment in ‘poly-plat’. SSP and SAP. This is three times the amount of lL-1a released by cisplatin-treated macrophages. TNF-a release TNF-a increases were observed in SSP (5 yg/ ml) at all times teSted, with the most significant increase occurring at 2 h post-treatment (3250 pg/ ml) (Figure 3). This level dramatically decreased at 24 h pose treatment (565 pg/ml). ‘Poly-plat' and SAP (5 [43/ ml) demonstrated very little TNF-a activity. barely reach- ing 200 pg/ml at 24 h post-treatment. Cisplatin demonstrated the usual enhanced release of TNF-a at various time intervals. reaching a peak value at cytoplasmic extensions radiate from the cell body in all directions. While SAP (5 ul/mlHreated macro- phages did not show any extension formation. but instead assumed a discoid shape similar to that of the normal macrophages (Figure 4C and D). Cisplao tin (5 ,ug/ml)-treated macrophages also showed simi- lar extension formation but only after 24 h of treatment. Lysosomal studies 2 h post—treatment (300 pg/ml). . Based on fluorescence measurements after acridine _ orange labeling. a SOO-fold increase in the number of Macrophage activation lysosomes in the macrophages after only 2 h of Murine peritoneal macrophages demonstrated exten- sion formations after 2 h of treatment with ‘poly- plat' and SSP (5 ug/ml) (Figure 4A and B). These 15 poly-plat‘ treatment (Figure 5A) was observed com- pared to normal cells (Figure 58). The lysosomes were plentiful in the cytoplasm of the macrophages and in the drug-induced cytoplasmic extensions Arm-Cancer Drugs - Vol 8 - I99” 325 Hj Muenchen et al. 4000? 3500'(’ 3000" TNF concentration (pg/ml) 0.5 1 I Normal I Cisplatin I Poly-plat D SSP I SAP - 2 24 Various drugs post-treatment (h) Figure 3. Bar graph showing TNF-a release in the supernatant of murine peritoneal macrophages treated with either cisplatin. ‘poly-plat‘, SSP or SAP (5 rig/ml) after 30 min. and 1. 2 and 24 h post-treatment. Note the maximum increase in TNF-a tor cisplatin and SSP 2 h post-treatment. Although. 'poly-plat’ and SAP did show an increased level of TNF-a they were not significant compared to cisplatin or SSP treatments. radiating from the cell body. SSP and SAP both demonstrated a loo-fold increase in the number of lysosomes. Comparatively. cisplatin treatment de- monstrated only a 50-fold increase in the lysosomes (Figure 5C), both in the cell body and the cytoplas- mic extensions. Discussion The anti-tumor agent cisplatin has been shown to enhance the immune system.‘2 Cisplatin activates the macrophages by inducing an increase in their cytoplasmic lysosomes and promoting extension formations that establish contact with the tumor cells.15 These macrophages lyse tumor cells through the transfer of lysosomes via the cytoplasmic exten- sions.” Cytolytic faCtors such as H302. superoxide anion. IL—la. TNF-a. lysozyme and flNl-l have been demonstrated to be released from these treated macrophages."” Our study. using the second gen- eration analogs of cisplatin. has shown that ‘pon- plat', SSP and SAP elicit much more IL-la. TNF-a. macrophage activation. as depicted by extension formations. and lysosomal increase compared to cisplatin. 326 Ami-Cancer Drugs - Vol 8- I‘ ‘ 16 Il-la release by aetivated macrophages and its cytotoxicity to tumor target cells proves it to be potent mediator in tumor cell killing by macro- phages.H Its release in vitro occurs in a cyclic manner showing its greateSt increase at 2 h post- treatment after ‘polyoplat‘. SSP and SAP. lL-la can deStroy tumor cells through several different path- ways. i.e. production of superoxide. nitric oxide and hydrogen peroxidc.'5~‘° resulting in lipid peroxi- dation.l mitochondrial membrane depolarimtion and calcium mobilization. reduction in ATP synthesis.” and DNA base alterations.l9 Compared to cisplatin treatment, where the lL-la release reaches a maximum at 24 h. these new drugs achieved it only after 60 min. reaching a maximum at 2 h. If the levels of IL-la are any indication of the cytotoxicity then the emciency of these drugs need to be tested in the in viva system. TNF-a also plays a regulatory role in inflammation and immunological response to tumors.20 TNF—a aetivates production of nitric oxide which induces iron loss. and inhibits DNA synthesis. mitochondrial respiration and the citric acid cycle.“22 It is clear that TNF-a is an important mediator of tumor cell death by macrophages. Of the three analogs tested. only SSP demonstrated a rise in TNF-a production. . V K _ ,2... . C .. g 1 I a £51.; ' " o . B I D - —.-:- Figure 4. Light micrographs showing macrophages at 24 h in normal medium (A). and after 2 h of ‘poly-plat' (B),. SSP (C) and SAP (D). Note the extension formation after ‘poty-plat‘ and SSP treatment(s) Only SAP assumes a discoid shape similar to that of untreated macrophages. Originalmagnification: x1600. Bar- - 0.5m Possibly the main thrust of SSP action is through increased release of TNF—a and lL-la early enough compared to cisplatin. Lysosomes take part in tumor cell death through macrophage activation? 2° Cisplatin-treated macro- phages show an increase in their number of lyso- somes and have been shown to be transferred to tumor cells through cytoplasmic extensions. This results in the lysis of tumor cells.2 A greater increase in lysosomes is seen in 'poly-plat’-. SSP- and SAP—treated macrophages when compared to cispla— tin-treated macrophages. lf lysosomal activity is any Platinum analogs and immune system indication of cytotoxicity then 'poly—plat' may have greater efficacy in tumor cell death. ‘Poly-plat‘ is curative in a range of solid tumors including renal. breast. ovarian. plasma cell myeloma and adenocarcinoma. It has been shown to be up to 15 times as active as equimolar amounts of cisplatin. showing inhibition in many tumors where cisplatin is ineffective: Recently. results have shown ‘poly-plat‘ to have higher potency than cisplatin at the same level in vitro and in viva. This is due to ‘poly-plat‘ having a molecular weight which is more than lO—fold higher than cisplatin. ‘Poly-plat’. which contains only one-fifth the platinum of cisplatin. has also been shown to be less toxic. SSP is curative of many cisplatin resistant tumors including M5 ovarian and several plasma cell myelo- mas. SSP also elicits no nephrotoxicity and is less toxic than cisplatin even at LDw dose (180 mg/kg). In vitro. SSP treatment resulted in the greatest increase in lL-la and TNF-a release. From our in m‘tro studies it seems that SAP. unlike cisplatin. 'poly-plat‘ or SSP, does not funcrion through the activation of the immune system. How- ever. in in vivo studies, it has been shown to be most potent in the regression of X5563 plasma cell myeloma. M-S ovarian carcinoma. 6C3HED lympho sarcoma. CSH mammary adenocarcinoma. 816 mela« noma and DLD-Z human colon tumor xenografts. Compared to cisplatin‘s 9% inhibition of the DLD-Z human colon tumor xenografts. SAP has demon- strated a 99% inhibition. Whether its mechanism of action is through increased macrophage lysosomal activity or more effeaive DNA cross-linking remains to be explored. Concluslon ‘Poly~plat‘. SSP and SAP induce murine macrophage activation via the production of cytoplasmic exten— sions. lysosomes. and cytolytic factors lL-la and Figure 5. Fluorescent images taken from the Zeiss 10 laser scanning confocal microscope of macrophages labeled with acridine orange (5 ug/ ml) showing lysosomal fluorescence in untreated (A). Cisplatin (B) and ‘pon-plat' (C). Note the large increase in lysosomal fluorescence after ‘poly-plat' treatment. Original magnification: X16000. Bar = 10 um. Ann-Canter Drugs ~ V018 » 199‘ 327 H] Muenchen et al. 'I'NF-a. Previous invesugations have shown these drugs to be more effective than cisplastin in vitro and in viva while eliciting less toxicity. Our studies support the activation of the immune system via the mediation of cytolytic factors and lysosomes as possible mechanisms of action of these drugs. Based on our observations we pr0pose that ‘poly-plat‘. SSP and SAP activate various cytolytic factors of the immune system better. compared to cisplatin. References I») 10. II. 328 . Rosenburg B. VanCamp K. Krigas T. Inhibition of cell division on E. coli by electrolysis production from a platinum eleCIrode. Nature 1965; 252: 698. . Nicolini M. Platinum and other metal coordination compounds in cancer chemotherapy. Boston: Marti- nus Nijhofi; I990. . Adams D. Hamilton T. Activation of macrophages for tumor cell kill: effector mechanisms and regulation. In Heppner GH. Fulton AM. eds. Macrophages and cancer. Boca Raton. FL: CRC Press 1988: 27. . Carleton S. Stevenson A. I-Iibbs J. EffeCtor mechanisms for macrophage-induced cytostasis and cytolysis of tumor cells. In: Heppner GH. Fulton AM. eds. Macro- phages and cancer. Boca Raton, FL: CRC Press 1988: 39. . Palma JP. Aggarwal SK Cisplatin and carboplatin mediated activation of murine peritoneal macrophages in vitro: production of interleukin-1 and tumor necro sis factor. Anti-Cancer Drugs 1995; 6: 1. . Walker E, Gale G. Methods of reduction of cisplatin nephrotoxicity. Ann Clin Lab Sci 1981; 11: 397. . Fiebig HH. Dress M, Ruhnau T, Misra HK. Andrulis P. Hendriks HR. 6821. a novel platinum complex with antitumor aCtivity in human renal and mammary xenografts. ProcAm Ass Cancer Res 1996; 37: 297. . Meager A. RIA. IRMA and ELISA assays for cytokines. In: Balkwill F. ed. Cytokr'nes: a practical approach. Oxford: Oxford University Press 1991. 299. . Genzyrne. Elisa kit for quantification of mouse interleukin-la. Cambridge. MA: Genzyrne Corp. 1994’: 12. Genzyme. ELISA kit for quantification of mouse tumor necrosis factona. Cambridge, MA: Genzyme Corp. 1993: 12. Poole A. The detection of lysosomes by vital staining with acridine orange. In: Dingle J. ed. lysosomes: a laboratory handbook. Amsterdam: Elsevier/North Anti-Cancer Drugs - Vol 8 - l 99" 18 13. 14. 15. 19. 20. 24. Holland Biomedical 19‘": 313. . Sodhi A. Singh S. Interaction between cisplatin treated macrophages and Dalton's lymphoma cells in vitro. Exp Cell Biol 1989: 56: 1. Palma JP. Aggarwal SK. Jiwa A. Murine macrophage activation after cisplatin or carboplatin treatment. Anti-Cancer Drugs 1992; 3: 665. Palma JP. Aggarwal SK. Cisplatin and carboplatin mediated release of cytolytic flaws in murine penro neal macrophages in vitro. Anti-Cancer Drugs 1994; 5: 615. Matsubara T. Ziff M. Increased superoxide artion release from human endothelial cells in response to cytokines.jlmmunol 1986; 137: 3295. . Zharazmi A, Neilsen. H. Bendtzen K. Recombinant interleukinol alfa and beta prime human monocytes superoxide production but have no effect on chemo- taxis and oxidative burst response of neutrophils. lmmunobiolog‘i' 1988; 177: 32. .. Arouma O. Halliwell B. Dizdaroglu M. Iron ion dependent modification of bases in DNA by super- oxide radical generating system hypoxanthrne xanthine oxidase. 1. Biol Chem 1989; 264: 13024. . Richter C. Kass G. Oxidative Stress in mitochondria: its relationship to cellular calcium homeosrasis. cell death. proliferation and differentiation. Chem-Biol Interact 1991: 77: 1. Chong Y. Heppner G. Paul L. et al. Macrophage mediated induction of DNA strand breaks in target cells. Cancer Res 1989: 49: 6652. Balkwill F, Naylor M. Malilt 8. Tumor necrosis factor as an anticancer agent. Eur] Cancer 1990; 26: 641. . Esumi I-I. Tannenbaum S. US—Japan Cooperative Can- cer Research Program: seminar on nitric oxide synthase and carcinogenesis. Meeting Report. Cancer Res 1994; 54: 29‘. . Knowles R. Moncada S. Nitric oxide synthases in mammals. Biocbem 1 1994; 298: 249 . Bucana C. Hoyer L. Hobbs B, et al. Morphological en'dence for translocation of lysosomal organelles from cytotoxic macrophages into the cytoplasm of tumor target cells. Cancer Res 1976; 36: 4444 Hrbbs J. Heterocytolysis by macrophages activated by bacillus calrnelte guerin: Iysosome exocytosis into tumor cells. Science 1974; I48: 468. . Singh S. Sodhi A. Interaction between cisplatin treated macrophages and Dalton‘s lymphoma cells. in vitro Exp Cell Biol 1989; 56: 1. (Received 11 February 1997’; accepted 18 February 1997) CHAPTER 4: INIMUNE SYSTEM ACTIVATION BY CISPLATIN AND ITS ANALOG “POLY—PLAT”: AN IN VITRO AND IN VIVO STUDY l9 Anti-Cancer Drugs 1998. 9. pp. 93-99 Research paper Immune system activation by cisplatin and its analog ‘poly—plat’: an in vitro and in vivo study Heather J Muenchen and Surinder K Aggarwal Department of Zoology. Michigan State University. East Lansing, MI 48824-1115. USA. Tel: (+1) 517 353-2253; Fax: (+1) 517 432-2789. Swiss Webster mice treated with bolus injections of ‘poly-plat' (10 mglltg) show increased activation after 2 and 12 days when isolated in cultures. Such macrophages demonstrate an increase in the number of lysosomes and cytoplasmic extension iormatlon with enhanced cytoltine (interleukin-11) activity. in addition, peripheral blood smears demonstrated an increase in lymphocytes and monocytes compared to cisplatin—treated animals. These results show that ‘poly-plat' activates the immunesystemmoreellectivelythanclspiatinbothlnvitro andlnvivclc 1998 RapidScienceLth Key words. Cisplatin. interleukin-1:, in vitro. in vivo. macrophages, nitric oxide, ‘poly-plat'. Introduction Regulation of normal and abnormal cell growrh is the primary function of the immune system.1 Many different immune system cells such as macrophages. lymphocytes and monocytes secrete various cytokines when aetivated by viruses. parasites. antigens or tumor cells.2 Macrophages are now being considered as one of the m05t p0tent components of the immune sy5tem actively involved in tumor regression and tumor cell lysis. Macrophages have the capacity to destroy tumor cells not only by direct contact and phagocytic activity but also by extracellular release of cytolytic factors that are capable of killing tumor cells.’ Cisplatin [als-dichlorodiammine—platinumam is a potent antitumor compound which has been success fully used against a number of animal"5 and human tumors.6 Although the primary target for cisplatin is the inhibition of DNA synthesis: it has also shown The authors wish to thank Andnrlis Pharmaceuticals (Beltsn’llc. MD) for their generous gift of poly-plat' and cisplatin used in these experiments. Correspondence to SK Aggarwal C l 998 Rapid Science Ltd 20 development of Specific cellular immune responses in tumor-bearing mice.8 Cisplatin has been shown to activate murine peritoneal macrophages in vitro.9 These activated macrophages seek out tumor cells through the formation of cytoplasmic extensions and lysosomal transfer to these target cells causing cell death.10 Cisplatin-treated macrophages have also been induced to release various cytolytic factors including interleukin (ID-1:1 and tumor necrosis factor (TNT-)4 which have potent antitumor activities.2 A combina- tion therapy. using cisplatin and immunostimulants. has demonstrated a synergisdc enhancement in its antitumor activity. Poly-[trans—l .2 - diaminocyclohexane)platinum] - car- boxyamylose (poly-plat’) is a second generation analog of cisplatin with higher efficacy and pctency. while exhibiting less toxicity.”12 ‘Poly-plat‘ is equi- potent to cisplatin with only one-fifth the platinum content.“2 The prospect of a new drug which is capable of enhancing the immune system with less severe side effects is very promising. Here we have further explored the effects of ‘poly-plat' in vitro and in vivo on the macrophage as an effector cell and its interaction with tumor target cell(s). Although its exact mechanism of aetion is not known. it has been shown to enhance the immune system b0th in vitro and in vivo more efl‘ectively than cisplatin.H Materials and methods Cell cultures Swiss Webster mice (Charles River. Wilmington. MA) were sacrificed by cervical dislocation and peritoneal macrophages were isolated by injection of 5 ml chilled minimal essential medium (MEM; Gibco. New York. NY) without serum containing 1% antibiotic-antimyv Ann-Cancer Drugs ~ Vol 9 - 1998 93 H] Muenchen and SK Aggarwal cotic (Gibco) [penicillin G (10000 U/ml). Streptomy- cin sulfate (10000 rig/ml) and amphotericin B (25 ug/ ml)] into the peritoneal cavity. After gently massaging the abdominal wall. cells were aspirated and seeded onto 18 mm2 glass coverslips. These were placed in 35 mm Petri dishes at 2-4 x 106 cells/ml and incubated for 2 h at 376C after which cells were wasth vigorously to remove non-adherent cells. Cell cultures were incubated in normal medium (minimal essential media and 10% heat-inactivated fetal calf serum) at 37°C in a 5% C02 incubator. Sarcoma 180 ascites ($180: CCRFSI8OII: ATCC, Rockville. MD) served as target cells for macrophages. Normal hepatocytes. obtained by mincing a small piece of liver through a fine wire mesh, 105 x 105 um in size (T etito, 11.). also served as target cells for the macrophages. An effectorztarget cell ratio of 1:10 was maintained in all experiments. Treatments in vitro ‘Poly-plat‘ (Andrulis Pharmaceuticals, Beltsville, MD) was prepared in 0.85% NaCl in 5 rig/ml concentra- tions. Macrophages were treated with the drugs for 2 h. The drug(s) containing medium was replaced by normal medium and supernatant (500 [11) was col. lected at 0.5. 1, 2, and 24 h for cytolytic factor(s) analysis. In addition. macrophages were also treated with cisplatin (5 rig/ml) dissolved in 0.85% NaCI with 3 til/ml of dimethyl sulfoxide (DMSO: Sigma, St Louis. MO). Untreated cells in normal medium served as controls. Interferon (IFN);'/lipopolysaccharide (LPS) (Signal Transduction laboratories, Lexirrgton, KY) was simultaneously added to some cultures at 0.8 rig/2 pl. Coverslips were fixed and stained to view the changes in structural morphology in terms of extension formation and lysosomal changes. Treatments in vivo Mice (20 g) were injected (i.p.) with ‘poly-plat’ (10 mg/kg) or cisplatin (10 mg/kg) with or without subsequent injections of IFN-y/IPS (0.8 rig/2 [1]). Normal mice were given i.p. injections of 0.85% NaCl to serve as controls. Mice were sacrificed after 2 and 12 days, and peritoneal macrophages were isolated. lL-ia assay lL-l-ar was assayed using ELISA kits (Genzyme, Carn- bridge, MA). The method used the multiple antibody sandwich principle. where monoclonal anti-murine II.- 94 Anti-Cancer Drugs - Vol 9 . I998 21 1a was used to bind murine IL-la present in the supernatant. A biounylated polyclonal antibody bind- ing the IL-11 was added and unbound material was washed out. Peroxidase-conjugated avidin was used to bind these biotin-tagged complexes. A substrate solution was then added resulting in a color change. The reaction was stopped by acidification and absorbance was read at 450 nm. Standard curves were generated with 11:11 (15—405 pg/ml) provided in the kits and linear regression analysis was performed. Macrophage tumor cell interaction studies in vitro Variously drug-treated macrophages were co-incu- bated with 5180 cells for 30 min. and 1. 2 and 24 h. Coverslips were fixed and stained to view any interactions between the effector and the 8180 target cells. Peripheral blood smears Peripheral blood smears were stained using Giemsa‘s stain” and cell counts were made of the lymphocytes. including monocytes. Nitrite assay for estimation of NO production The concenu-ation of stable nitrite, end product from N0 oxidation by effector macrophages. was deter- mined by the method of Ding et al.H using Griess reagent. Briefly, 100 ll] of supernatant from untreated and treated macrophages collected at various times was mixed with equal volume of Griess reagent (1% sulfanilamide. 5% phosphoric acid. 0.1% naphthylethy— Ienediamine dihydrochloride; Sigma). The mixtures were incubated for 10 min at room temperature and the absorbance read at 540 nm. Standard curves were generated using 1 nM to 220 M NaNOz and nitrite concentrations were determined using linear regres sion analysis. Inducible NO synthase (iNOS) staining Macrophage monolayers were stained for iNOS which catalyzes the oxidation of L-arginine to citrulline and NO using the avidin-biOtin-peroxidase complex method.” iNOS was confirmed by the Vectastain Elite ABC Kit (Vecror. Burlingame. CA). Results lL-1ar rebase There was observed a many fold increase in lL-la levels in the supernatants of macrophages in vitro after ‘pon-plat' (10 trig/kg) treatment for various time intervals (Figure 1) compared to cisplatin treatment. Similarly. macrophages isolated from ‘poly-plat'treated mice demonstrated a significantly higher level of IL-la (500 pg/ml) than cisplatin treatment (250 IDS/ml). but only after 12 days of treatment (Figure 2). After 2 days of ‘poly-plat' treatments the levels of the IL-lar were close to normal. However. after 2 days of ‘poly-plat' treatment there was an increase in the formation of cytoplasmic extensions and the lysosomes (Figure 3A) compared to cisplatin treatment (Figure 3B). When ‘poly-plat’- or cisplatin-treated mice were injected with IFN-a/IPS .(0.8 rig/2 141) there was a significant decrease in the levels of 11:11 for both the drug treatments (Figure 4). However, there was a significant induction of cytoplasmic extensions and the number of lysosomes (Figures 5A and B). Macrophage-tumor cell interaction studies in vitro Normal murine peritoneal macrophages. when trea- ted with ‘poly-plat’, show cytoplasmic extensions which radiate from the cell body after only 2 h post. 400 v i _l 350 ‘ I Normal 300 .. I Cisplatin fi D 'Poiy-plat' \ 250 1' or 3 . 3 200 r =3 150 ‘- 100 t 50 ll 0 —-'D—-— ' 0.5 1 2 24 Post-treatment (h) Figure 1. Bar graph showing lL-1: in the supematants of cultured murine peritoneal macrophages treated with either ‘poly—plat’ or cisplatin (5 rig/ml) alter 30min. and 1, 2 and 24 h post-treatment. Note a sudden increase after 1h of ‘poly-plat’ treatment compared to cisplatin. This increase stays at the same level through 2411 post-treatment. Cisplatin treatment induces a gradual increase through 24h but is still less than that of ‘poly-plat'. 22 ‘Poly-plat' and the immune system treatment. When these drug-treated macrophages are co-incubated with $180 tumor cells they immediately establish contact with several target cells and form cytoplasmic continuity through which lysosomes are transported into the tumor cells causing their lysis (Figure 6). Cisplatin-treated cells also form cytoplas- mic extensions that are fewer in number and establish contact with fewer tumor cells compared to ‘pon-plat’treated macrophages. Normal macro phages do not form cytoplasmic extensions and when co-incubated with 8180 cells, fail to show any interaction. Peripheral blood smears Mice treated with ‘poly-plat’ demonstrated a 10-fold increase in the number of lymphocytes and monocytes both after 2 and 12 days post-treatment. Compan- tively, cisplatin-treated mice demonsuated a significant decrease. Nitrite assay for estimation of NO production Treatment of macrophages with ‘poly-plat' or cisplatin demonStrated an increase in the nitrite levels. as early as 30 min. that gradually dropped down to normal after 24 h of treatment (Figure 7). No distinct pattern was seen when comparing the relative increase in NO 500 r 450 “ I Normal i I I Cisplatin I 3501. : 'Poly-plat' lL-lrx lpg/ mll w i 1501’ A r 12 Post-treatment (days) Figurez. Bargraphshowing lL-1areleaseintl'resmer- natants of murine peritoneal macrophages from mice treated with either ‘poly-plat’ or cisplatin (5 119/111.) after 2 and 12 days. Note the large increase in lL-i: at 12 days post- injection with ‘poly-plat-treated mice. This increase is more than twice that of cisplatin-treated mice. Anti-Cancer Drugs ‘ Vol 9 - I998 95 H] Muenchen and SK Aggarwal Figure 3. Light micrographs showing the murine peritoneal macrophages of mice treated with ‘poiy-plat‘ (A) and cisplatinWS (B) alteerays. Notetheverylargenuclei nu (arrowheads)and long cytoplasm extensions(arro memos lysosomes radiating from the macrophages of p'oly-plaf-treated mice. There is very little of this activation olthe macrophages from cisplatin-treated mice. Bar=25 urn. I Normal+lFN-nyP$ I CisplatimlFN-y/LPS I 'Poly~plat'+lFN-y/LP$ lL‘lrr (pg/ mll 8 8 8 8 Post-treatment (days) Figure4. Bargraphshowrng IL-1zreleaseinthesuper- natantsotmurineperitonealmacrophagesirommicetreated with either poty- plat' or cisplatin (Sag/ml) plus IFN-, /LPS alter2and12days. Notethein malincreaseol lL-11afler2 daysbutasubsequentdecreasealtertZdaysol‘poly-plat' andcisplatintreatmentreachingclosetonormal levels between ‘poly-plat'- and cisplatin-treated macro pluges at the various times tasted. iNOS-staining When macrophages were treated with 'poly-plat‘ or cisplatin, a lOfold increase in the expression of iNOS was visible after only 24 h post-treatment. while none was seen in untreated macrophages. A 20-fold increase 96 Anu‘Cancer Drugs - Vol 9 - 1998 23 in the expression of iNOS was observed in macro- phages treated with 'polyoplat‘ or cisplatin plus IFN-y/ LPS. Discussion Macrophages have been implicated in the destruction of tumor cells either by direct cell—cell contact or cell cytotoxic mechanisms via the release of extracellular mediators. ‘6 '- Poly'-plat and cisplatin have both been demonstrated to be such enhancers of the immune system in vivo and in virmf” ” w '9 These chemother- apeutic agents have been shown to activate murine peritoneal macrophages in a multiset fashion. increas ing their ability to recognize tumor cells, establish cell- cell contact. transfer lysosomes and induce tumor cell “55.10"“ Activated macrophages are known to release many cytolytic factors. including mum“ IL]: was first known to be a lymphocyte activating factor because of its ability to stimulate chlls.22 11-1: ean destroy tumor cells via the production of superoxide. NO and hydrogen peroxide.2124 resulting in lipid peroxida- tion.” mitochondrial membrane depolarintion and calcium mobilintion. reduction in ATP synthesis,36 and DNA base alterations}- in the present study we have observed not only an increase in lL-la but also an increase in monocytes and lymphocytes. in the peripheral blood. N0 is a noxious free radical reactive nitrogen intermediate (RNI) gas found to play a major role in various biological processes.” it has been estab lished as a messenger molecule regulating immune ‘Poly-plat' and tbe immune system Figure 5. Light microphages showing the murine peritoneal macrophages of mice treated (2 and 12 days) with poly-plat' (A) and and cisplatin (B) plus IFN-~,-/LPS after 2 days. Note the large nuclei, numerous macrophages of both ‘poly-plat’ and cisplatin treated mice. Bar=25 um. (arrows) radiating from the Figure 6. Light macrophages showing poly-plat' -treated macrophages co-cultured with 3180 cells for only 2h. Note the cytoplasmic extensions in contact with tumor cells (arrows) and an increase in the lysoscrnal activity only after drug treatments. Untreated macrophages never establish contactwithtumorcellsnorstmanyincrease in the number of lysosomes. Bar=25 urn. functions. blood vessel dilation as well as neuro- transmission. NO is formed through the stepwise oxidation of the guanidinonitrogen terminal atom of L-argininc to N0 24 long cytoplasmic extensions 120 I ‘ I Normal 100 I Cisplatin ._ Poly-plat' 4O Nitrite concentraction (nM) 20 0.5 1 2 2t Post-treatment (h) Figure 7. Bar graph demonstrating nitrite levels in the supematants of macrophages cultures alter treatment with ‘pon-plat’ or cisplatin at various time intervals. Note the intial increase in the nitrite levels at 30min. but dropping to normal levels after 24 h. and bcitrullinc catalyzed by M05.29 NO is further oxidized to form nitrite. NO exists in several forms. The constitutive form is found in endothelial cells and neurons of the central nervous system while the inducible form (iNOS) is present in macrophages. leukocytes and vascular smooth muscle cells. iNOS is induced by a variety of factors including endotoxins (cg. LPS) and cytokines (ll-la. lFN-y and TNF—a).5° Results show that ‘poly-plat' and cisplatin both influence the expression of iNOS and the observed increase in nitrite levels in the medium. It is not surprising to find an increase in NO levels after ‘poly-plat‘ and cisplatin treatment of the macrophages and mice. since both drugs have also been demon- strated to induce lL—lar release in the medium. Ann£mrrer Drugs - lb! 9 - 1998 97 H] Muenchen and SK Aggarwal NO released by primed macrophages mediates its cytOtoxic effecrs through loss of iron.” '52 inhibition of DNA syntl'tesisfz'55 mitochondrial respiration”’” and the citric acid cycle.35 These have all been attributed to the inactivation of ribonucleotidc reduCtase and the inhibition of iron-sulfur proteins NADH:ubiquinone oxidoreductase. NADstuccinate oxidoreductase and mitochondrial aconitase. In addition. the reactive oxygen species generated by macrophages could combine with NO to form substances that are more potent than NO itself.” NO combined with super- oxides could yield peroxynitn’te that decomposes to the hydroxide free radical and N02 free radical. Our results demonstrate an immediate increase in nitrite concentration, in ‘poly-plat'- and cisplatin-treated macrophages, after 30 min in culture. These processes may attribute to the cytotoxic ability of 'poly-plat‘- or cisplatin-primed macrophages inducing the destruc- tion of tumor cells. Conclusion The results of this study strongly suggest the enhancement of the immune system by the anticancer agent 'poly-plat'. In addition to inducing direct cell- ccll contact of the macrophages with the tumor cells. there is a release of various cytolytic faCtors (IL-lat and NO) from such macrophages. Release of these factors. shown to inhibit tumor cell proliferation. is evidence for the role they play in ‘poly-plat'-mediated tumor ccU toxicity. The major mechanism of action of ‘poly-plat‘ is still unknown. However, our studies support the hypothesis that the enhancement of the immune system is an important mechanism of action of ‘poly— plat'. Based on these findings. we propose that ‘polyo plat‘, macrophage-mediated cell cytotoxicity. involves various cytolytic factors including 11-11 and NO. The activation of macrophages. a multi-step process. leads to target cell destruction through lysis. References l. Aggarwal B. Totpal K. Mechanisms of regulation of cell gowth by tytokines of the immune system. In: Pasquier C. Olivier R Auclair C. eds. Oxidative stress. cell activation and viral infection. Basel: Birkhauscr Verlag 1994: 155. . Palma J. Aggarwal S. Cisplatin and carboplatin mediated activation of murine peritoneal macrophages in vitro: production of interleukin-1: and tumor necrosis factor-z. AntrLCancer Drugs 1995; 6: 1. 3. Sodhi A Singh 8. Release of cytolytic faCtorts) by murine macrophages in vitro on treatment with cisplatin. Int 1 lmmunopbarmacol 1986; 8: 701. N 98 Anti-Cancer Drugs - Vol 9 . I 998 U! 10. ll. 13. 14. . Ilch P. Wolf W. Platinum complexes: a new class of anti- neoplastic agents. ] Pbarmacol Sci 1976: 65: 315. . Rosenburg B. \‘anCamp L The successful regression of large solid sarcoma 180 tumors by platinum compounds. Cancer Res 19‘0: 30: 1799. . Durant J. Cisplatin: a clinical overview. in: Prestayko A. Crook: S. Carter S. eds. Cisplatin: current status and new detelopments. New York: Academic Press 1980: 517'. . Zwelling L Kohn K. Mechanism of action of cis- dichlorodiammine platinum (II). Cancer Treat Rep 19‘9; 63: 1459. . Bahadur A Santa S. Sodhi A Enhanced cell mediated immunity in mice after cisplatin treatment. Polish 1 Pbamtac Pharmacy 1984; 36: ‘HI. . Singh S. Sodhi A Interacuon between Cisplatin treated macrophages and Dalton's lymphoma cells in vitro. Exp Cell 81011989156: 1. Palma J. Aggarwal S. Jiwa A. Murine macrophage activation after cisplatin or carboplatin treatment. Anti- Cancer Drugs 1992; 3: 665. Muenchen H. Aggarwal S. Misra H. Andrulis P. Enhanced immunostimulation by novel platinum anticancer agents. AntiCancer Drugs 199': 8: 323. . Fiebig H. Dress M. Ruhnau T. Misra H. Andrulis P. Hendriks H. G821. a novel platinum complex with antitumor activity in human renal and mamman xeno grafts. MA»: .453 Cancer Res I990: 37: 29". Kiernan J. Histological €- birtocbemr‘cal methods. 0x- ford: Pergamon Press. 1990. Ding A. Nathan C. Stuehr D. Release of metive nitrogen intermediates from mouse peritoneal macro- phages: comparison of activating cytoldnes and evi- dences for independent production. 1 Immunol 1988: 141: 240’. . Hsu S. Raine L Fangcr H. Use of avidin-biotin- peroxidasc complex (A85) in immunoperoxidase tech nique: a comparison between ABC and unlabeled antibody (PAP) procedures 1 Hr’stocbem Cytochem 1981: 29: 5“; . Adams D. Hamilton T. Activation of macrophages for tumor cell kill: effeCtor mechanisms and regulation. In: Heppner GH. Fulton AM. eds. Macrophages and cancer. Boa Raton. FL: CRC Press 1988: 2'. . Stewart C. Stevenson A. Hibbs J. Effector mechanisms for macrophage-induced cytosmsis and cytolysis of tumor cells. In; Heppner GH. Fulton AM. eds. Macrophages and cancer. Boa Raton. FL' CRC Press 1988: 39. . Sodhi A. Singh S. Interaction between cisplatin treated macrophages and Dalton‘s lymphoma cells in vitro. £5.11) Cell Biol 1989: 56: l. . Palma JP. Aggarwal SK. Cisplatin and carboplatin mediated release of cytolytic factors in murine peritoneal macrophages in vitro. Anti-Cancer Dmgs‘ I994; 5: 615. . Lachman L et al. Natural and recombinant interleukin-ll? is cytocidal for human melanoma cells. 1 Immunol 1986: 136: 5098. . Okubo A Sone S. Tanaka M. et al. Membrane associated interleukin-la as a mediator of tumor cell killing by human blood monocytes fixed with paralonnaldehydc. Cancer Res 1989; 49: 256. . Gery l. Waksman B. Potentiation of cultured mouse thymocyte response by factors released by pcnpheral leukocytes. _] Immunology 1986: 107: 1"‘8 26. . Matsubara T. Zifi‘ M. Increased superoxide anion release from human endothelial cells in response to qtokines. j Immunol 1986: 137: 3295. ’. Kharazmi A, Neilson H, Bendtzen K. Recombinant interleukin-1:: and B prime human monocytes super— oxide but have no effect on chemotaxis and oxidativc burst response of neutrophils. Immunobr'ology 1988; 177: 32. . Arouma O. Halliwell B. Dizdaroglu M. Iron ion dependent modification of bases in DNA by superoxide radical generating sysmm hypoxanthine oxidase. ] Biol Chem 1989: 264: 13024. Richter C. Kass G. Oxidative stress in mitochondria: its relationship to cellular calcium homeostasis. cell death. proliferation and differentiation. Cbem-Biol Interact 1991; 77: l. . Chong Y. Heppner G. Paul L et al. Macrophage mediated induCtion of DNA and breaks in target cells. Cancer Res 1989: 49: 6652. Lowenstein C. Synder S. Nitric oxide. a novel biolog'c messenger. Cell 1992: 70: 705. . Moncada S. Palmer R. Higgs E. Nitric oxide: physiology. pathophysiology, and pharmacology. Pbarmacol Rev 1991: 43: 109. 26 35. ‘Poly-plat’ and the immune system . Esumi H. Tannenbaum 5. Seminar on nitric oxide synthase and carcinogenesis. Cancer Res 1994: 54: 29‘. . Hibbs j. Taintor R Vavrin Z- Macrophage Q'totoxiu'tv: role for L-arginine daminase and amino nitrogen oxidation to nitrite. Science 1984: 235: 4‘5. . Stuer D. Nathan C. Nitric oxide. A macrophage producr responsible for cytosrasis and respiratory inhibition in tumor target «:15an19 Med 1989: 169: 1545. . Krahenbuhl 1. Remington J. The role of activated macrophages in specific and nonspecific cytostasis of tumor cells] Immunol 1974; 113: 507'. . Granger D. lehninger A. Site of inhibition of mitochon- drial electron transport in macrophage-minted neOplasuc cells. J Cell Biol 1982: 95: 521’. Drapier J. Hibbs J. Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-argininedependent inhibition of mitochondrial iron~ sulfar enzymes in the macrophage efl‘ector cells 1 Immunol 1988; 140: 2829. (Received 23 September 199 7: accepted 3 October 199 7) 99 Arm-Cancer Drugs - Vol 9 - I 998 CHAPTER 5: ENHANCED IMMUNE SYSTEM ACTIVATION AFTER TREATMENT WITH NOVEL ANTINEOPLASTIC AGENTS 27 ANTICANCER RESEARCH 15; 2631-2030 (1998) Enhanced Immune System Activation after Treatment with Novel Antineoplastic Platinum Agents HEATHER J. MUENCHEN and SURINDER K AGGARWAL Department of Zoology. Michigan State University. East Lansing. MI 48824-1115. USA. Abstract. “Poly-plat". SSP. and SAP are second generation analogs of cisplatin ( C DDP) with higher efficacy and potency. In order to understand the mechanism of action of these compounds. isolated munne peritoneal macrophages were treated wrth "poly-plat". SSP. or SAP (5 ug/ml) for 2 h. Treated macrophages demonstrated an increase in the number of lysosomes. but only "poly-plat" and SSP treated macrophages were stimulated to form the cytoplasmic extensions so very characteristic of cisplatin after 2 h and 24 h post-treatment. SAP showed cytoplasmic extensions only after 24 h post-treatment. and demonstrated a back to the normal discoid form when viewed at 24 h post-treatment. When drug treated macrophages were co-incubated with S 180 tumor cells. cytoplasmic extensions of the macrophages developed contacts. and cytoplasmic continuity with the tumor cells, and a subsequent transfer of lysosomes from macrophage to tumor cell was observed after only 2 h of co-incubation. After 24 h of cocincubation, lysis of 5180 cells was acheived. Analysis of the tissue culture supematants collected from “poly-plat", SSP. and SAP treated macrophages demonstrated the enhanced activity of interleukin- la of over 400 pg/ml after 2 h post-treatment. compared to only 300 pg/ml with cisplatin 24 h post-treatment. However. only SSP demonstrated an increase in TNF-a activity (2000 ngl) after 2 h post-treatment. which is comparable to that of cisplatin. Based on our observations we propose that “poly-plat”. SSP. and S4P activate various cytolytic factors of the immune system better, than cisplatin. Cisplatin [cis-diamminedichloroplatinum (II): CDDP] is an antineoplastic drug with demonstrated activity against ovarian Correspondence to: Dr. Surinder K Aggarwal, Department of Zoology. Michigan State University. East Lansing. Ml 48824- 1115. Phone 517 3532253. Fax 517 4322789. e-mail l9813SKK@msu.edu Key Words: Cisplatin. “Poly-plat". SSP. SAP. macrophages. interleukin-la. tumor necrosis factor-a. lysosomes. in vitro. in viva. 0250-7005/98 $100+ .40 28 and testicular cancers”). DNA denaturation is one of the accepted methods of its mechanism of action through its intrastrand and interstrand cross-links interfering with DNA replication and transcription(2). Cisplatin has also been shown to enhance specific cellular immune responses in tumor bearing micc(3). through activation of murine peritoneal macrophages in vitro and in l‘ll'0(4.5). These activated macrophages seek out tumor cells through the formation of cytoplasmic extensions and lysosomal transfer to the target cells causing their death(6). Activated macrophages have been found to effectively destroy target cells by cytotoxic mechanisms(7.8). The activation process includes the generation of extracellular products including interleukin-la (IL-1a). tumor necrosis factonu (TNFoa) and nitric oxide (NO)(5.9). Although cisplatin is an effective chemo- therapeutic drug. it has very severe toxic side effects. of which gastrointestinal and nephrotoxicity are the dose limiting factors( 10). Poly-[(trans-l.2.diaminocyclohexanc) platinuml-carboxy- amylosc (”poly-plat"). S- sulfosalicylatmns-(1.2-diamino- cyclohcxane) platinum (SSP). and 4-hydroxy-(r-sulfo- nylphenylacetato (trans-1.2-diaminocyclohexane) platinum (11) (SAP) are analogs of cisplatin with higher efficacy and potency. This is especially true of “poly-plat" which contains 1/5 the platinum of cisplatin(ll). The prospect of new drugs which are capable of enhancing the immune system with less severe side effects is very appealing. and thus this investigation of “poly-plat". SSP. and SAP as macrophage activators was undertaken. Materials and Methods Cell Cultures. Swiss webster mice (Charles River Laboratories. MA) were sacrificed by cemcal dislocation and peritoneal macrophages were isolated by injection of 5 ml chilled minimal essential medium (MliM; Giboo. NY) without serum containing 1‘»? antrhmtnc-anumycotic [penicillin G (10 000 U/ml). strcptomycrn sulfate (lU mu rig. ml). and amphotericrn B (25 ugtmlll into the peritoneal cavrry After gently massaging the abdominal wall. cells were aspirated and seeded onto 18 mm’ glass coverslips. placed in 35 mm petri dishes. at 2-4 x 10" cellsxml and incubated for 2 h at 37'C. Cells were washed ngorously to remove 2631 ANTICANCER RESEARCH 18: 2031-2630 (1098) non-adherent cells. Cell cultures were inubaied in normal medium (minimal essential media and 10‘? heat inactivated fetal calf serum) at MT in a 5‘3? CO; incubator. Sarcoma ISO ascites (5180; CCRFS-lSOll; American Type Culture Collection. MD) served as target cells for macrophages. Normal hepatocytes. obtained by mincrng a small piece of liver through a fine wire mesh. 105 X 105 um in size (Tetlto. lnc.. lL). also served as target cells for the macrophages. An effector target cell ratio of MO was maintained in all experiments. Treatments. “Poly-plat" was prepared in 085‘} NaCl while. SSP and SAP (Figure l) were dissolved in 0.85% NaCl and 0.1“? NaCOa at S ugml. Macrophages were treated with the drugs tor 2 h. The drug containing medium was replaced by normal medium and supernatant (500 til) was collected at 0.5. l. 2. and 24 h for cytolytic factor analysis. in addition. macrophages were also treated with Cisplatin [5 iime dissolved in physiological saline with 3 ul'ml of dimethylsulfoxide (DMSO; Sigma. St. Louis. MO)]. Untreated cells in normal medium served as controls. Lysosomal assay. The quantitation of lysosomes before and after various treatments was achieved by exposing macrophage cultures to fresh medium containing acridine orange (5 rig/ml) for 30 min at 37‘C in the dark(12). After careful washing macrophages were examined under Zeiss 10 Laser Scanning Confocal Microscope. TNF-a assay. TNF-a released from supematants of the macrophages was assayed using specific analysis kits (Genzyme; Cambridge. MA). Again. the multiple antibody sandwich princrple was utilized with a murine monoclonal antibody specific for murine TNF-u in the samples(13). An HRP conjugated anti-murine 'l'NF-ci antibody was used to bind the multiple epitopes on TNF-o. A substrate solution was then added resulting in a color change. The reaction was stopped by acidification and absorbance was read at 450 nm. Standard curves were generated with TNF-a (35-2240 pg/ml) provided in the kits. and linear regression analysis was performed. IL-a assay. [bid was assayed using ELISA kits (Genzyrnc: Cambridge. MA). The method used the multiple antibody sandwich prtnciple(l3). where monoclonal anti-murine lL-la was used to bind murine lL-lo present in the supernatant. A biotinylated polyclonal antibody binding the lL-lo was added and unbound material was washed out. Peroxidme- conjugated avidin was used to bind these biotin tagged complexes. A substrate solution was then added. resulting in a color change. The reaction was stopped by acidification and absorbance was read at 450nm. Standard curves were generated with lL-lo (IS-405 pg/ml) provided in the kits and linear regression analysis was performed. Inducible nitric oxide synthase (iNOS) staining. Macrophage monolayers were stained for the inducible enzyme which catalyzes the oxidation of barginine to citrulline and NO using avidin-bioun~peroxidase complex method(l4). iNOS was confirmed by VECTASTAIN elite ABC Kit (Vector Laboratories, Inc.. Bullingame. CA). Nitrite assay for estimanon of nitric and: production The concentration of stable nitrite. the end of nitric oxide generation by effector macrophages. was determined by the method of Ding et al(15 ) based on Greiss reaction. Briefly. 100 rd of supernatant from untreated and treated macrophages collected at various times were mixed with equal volume of Greiss reagent (1% sulfanilamide. 5% phosphoric acid. 0.1% naphthylethylenediamine dihydrochloride; Sigma. MO). The mixtures were incubated for 10 min at room temperature and the absorbance read at 540 nm. Standard curves were generated using I nM-220 uM NaNO; and nitrite concentrations were determined using linear regression analysis. 2632 29 cl, A. I ‘I A. I A‘ (II) V ”LY-HA1“ .3. or, at a; —\ zit—o . \r.- '°"'\.... :93. ,4, / \ a,- ll- ..b-lu' . Al - n L a a -a a A a up. , u t v —‘ a a SSP I?~~~©~~ 4.0qu u- I.)- W) I“ 0') Figure l. Structures of various platinum coordination complexes studied. Results Macrophage activation. Normal murine peritoneal macrophages. when treated with “poly-plat" and SSP (5 tig/ml) for 2 h at 37'C in culture. developed extension formations within 2 h (Figure 2A and 23). These cytoplasmic extensions radiated from the cell body in all directions. While SAP (5 ug/ml) treated macrophages. did not show any extension formation. instead assumed a discoid shape similar to that of the normal macrophages (Figure 2C and 2D). Cisplatin (5 ug/ml) treated macrophages also showed similar extension formation but only after 24 h of treatment. Muenchen and Aggarwal: Immune System and Platinum Analogs C h Figure 2. Light micrograph: showing macrophages at 24 h in normal medium (AI: and after 2 h of “poly-plot" (B): SSP (CI. and SAP (DI Note die extension formation (ml after “'poiy-plat and SSP treament/s. while SAP treated macrophages. X1600, Bar=llhirn Macrophage tumor cell interaction studies. When the drug treated macrophages were co-incubated with S 180 tumor cells they immediately established contact with several target cells and formed cytoplasmic continuity through which lysosomes were transported into the tumor cells causing their lysis (Figure 3). CDDP treated cells also developed cytoplasmic extensions that were fewer in number and established contact with fewer tumor cells when compared to “poly-plat” treated macrophages. Normal macrophages did not form cytoplasmic extensions and, when co-incubated with 5180 cells, failed to show any interaction. Lysosomal studies Based on fluorescence measurements after acridine orange labeling. we observed a 500 fold increase in the number of lysowmes in the macrophages after only 2 h of “poly-plat' treatment (Figure 4A) compared to normal cells (Figure 43). The lysommes were plentiful in the cytoplasm of the macrophages and in the drug-induced cytoplasmic extensions radiating from the cell body, SSP and SAP both demonstrated a 100 fold increase in the number of lysosomes. Comparatively, cisplatin treatment demonstrated only a 50 30 rinoppearnncerothoseofunrmred rrticrogruphshowing" plat' treatedrruirrne peritorteol macrophagaco-culturedwithSlwcelLtforonlyZhNoumecytoplmlic thmdflihmmorcelblomnndonincreasednumba lysosornaX1600,8ar-losim Figure3 Light 2633 ANTICANCER RESEARCH 18: 2631-2636 (1908) Figure 4. Fluorescent images of macrophages labeled with acndtne orange (5 tigrnl) showing lysosomal fluorescence after “pith-plat " (A ;. rmrma.’ IE I. and Cisplatin (C I treatments. Note the large increase or Momma/fluorescence (arrows I after “putt-plat" treatment. X1000. Bar: lOum u i I It “Cy—t—N Figure 5. Gmph showing INF-a release in the supernatant of munne pentoneal macrophages treated mth either cisplatin. "poly-plat". SSP. ar SAP (5 ug/mlt after 30 min. 2 and 24 h post-treatment. Note the maximum increase in TNF-a for Cisplatin and SSP 2 h post-treatment. flough. ‘polv-plat" and SAP did show on increased level of TNF-a they were not Significant compared to Cisplatin or SSP treatments. fold increase in the lysosomes (Figure 4C). both in the cell body and the cytoplasmic extensions. INF-a release. Significant increases in TNF-o levels were observed in the supematants from SSP (5 pig/ml) treated macrophages at all times tested, with the most significant increase occurring at 2 h post-treatment (3250 pg/ml) (Figure 5). This level dramatically decreased at 24 h post-treatment (565 pg/ml). “Poly-plat” and SAP (5 pglml) demonstrated very little TNF-a activity. barely reaching 200 pg/ml at 24 h post-treatment. Cisplatin demonstrated the usual enhanced release of TNFoa at various time intervals. reaching a peak value at 2 h post-treatment (3000 pg/ml). IL-Ia release. Compared to cisplatin treatments there was an increase in IL-lo levels in the supematants of macrophages treated with either “poly-plat”, SSP, or SAP (5 ug/ml) for up to 24 h of testing (Figure 6). The greatest increases were seen 2 h post-treatment (400-500 pg/ml) with a subsequent 2634 31 mum-II) Figure 6. Graph thawing lL-la release in the supematants of munne penmneal macrophages treated wrth either (Uplaflrl. "rum-plat SSP. or SAP (jug ml; otter 30 mm. 2 and 24 h port-treatment. Note the lame increase at 2 h port-treatment tn "poly-plat". SSP. and S4P This ts three times the amount of IL-la released In t‘tsplottrt treated macrophages. decrease from there on. lL-lu levels demonstrated a consistent increase after cisplatin treatment. reaching a maximum after 24 b. However. lL-la levels. after “poly-plat". SSP. or SAP treatment. demonstrated a decline after a peak at 2 h post-treatment but these levels were still equal to or above those after cisplatin treatment. Inducible nitric oxide synthase (iNOS I staining. thn macrophages were treated with “poly-plat". SSP. or SAP expression of iNOS was visible while none was detected in untreated macrophages. Only moderate levels of iNOS were visible in cisplatin treated macrophages. Nitrite arsov for estimation of nitric oxide production Treatment of macrophages with “poly-plat". SSP. or SAP demonstrated increases in NO levels detected in cell culture supematants at varioustimes'lheutcreasewasseenasearlyaswmmmer treatment and persisted up to 24 h post-treatment. Again. cisplatin only demonstrated a moderate level of NO production. Muenchen and Aggarwal: Immune System and Platinum Analogs Discussion Cisplatin is able to activate peritoneal macrophages(4) in vitro and cause lysis of tumor cells(6). Cisplatin activates the macrophages by inducing an increase in their cytoplasmic lysosomes and promoting extension formations that establish contact with the tumor cells(6). These macrophages lyse tumor cells through the transfer of lysosomes via the cytoplasmic extensions(6). “Poly-plat”. SSP. and SAP. analogs of cisplatin. also have the capability to activate macrophages in vitro. Our study has shown that “poly-plat". SSP and SAP elicit much more macrophage activation, as depicted by extension formations, lysosomal increases. TNF-a. IL—la. iNOS. and NO compared to cisplatin. Lysosomes take part in tumor cell death through macrophage activation(16.17). A greater increase in lysosomes is seen in “poly-plat”, SSP and SAP treated macrophages when compared to cisplatin treated macrophages. If lysosomal activity is any indication of cytotoxicity the “poly-plat” may have greater efficacy in tumor cell death. TNF-a also plays a regulatory role in inflammation and immunological response to tumors(18). TNF-a activates production of nitric oxide which induces iron loss. and inhibits DNA synthesis. mitochondrial respiration and the citric acid cycle(19.20). Of the three analogs tested only SSP demonstrated a rise in the TNP-a production. Possibly the main thrust of SSP action is through increased release of TNF-a and IL-la early. compared to cisplatin. IL-la release by activated macrophages and its cytotoxicity to tumor target cells proves it as a potent mediator in tumor cell killing by macrophages(21). Its release in vitro occurs in a cyclic manner showing its greatest increase at 2 h post-treatment with “poly-plat". SSP and SAP. Compared to cisplatin treatment. where the IL-la release reaches a maximum at 24 h. these new drugs achieved it after only 60 min. reaching a maximum at 2 h. If the level of IL-la is any indication of cytotoxicity then “poly- plat” appears to be more efficient. The iNOS is induced by a variety of factors including endotoxins (cg. LPS) and cytokines (IL-la. IFN-y and TNF- a)(19). Results show that “poly-plat”. SSP. and SAP all demonstrated enhanced levels of iNOS. NO released by primed macrophages mediates its cytotoxic effects through loss of iron(22.23). inhibition of DNA synthesis(23.24). mitochondrial respiration(23.25) and the citric acid cycle(26). In addition. the reactive oxygen species generated by macrophages could combine with NO to form substances that are more potent than NO itself(27). NO combined with superoxides could yield peroxynitrite that decomposes to hydroxide free radical and N03 free radical. Our results demonstrate an immediate increase in nitrite concentration. in “poly-plat". SSP. and SAP treated macrophages. after 30 min in culture. These processes may be attributed to the cytotoxic ability of “poly-plat". SSP or SAP primed macrophages inducing the destruction of tumor cells. 32 Acknowledgements The authors wish to thank Andrulis Pharmaceuticals (Beltsville. MD) for their generous gift of “poly-plat”. SSP, SAP, and cisplatin used in these experiments. References l Litterst C: Cancer management in Man: Biological Response Modifiers. Chemotherapy. Antibiotics. Hyperthermia. Supporting Measures. In: Woolley P. ed. Platinum Compounds: Netherlands: Kluwer Academic Publishers; 1989: 85. Roberts .1. Pascoe .l: Cross-linking of complementary strands of DNA in mammalian cells by antitumor platinum compounds. Nature 235: 282. 1972. 3 Bahadur A. Sarna S. Sodhi A: Interaction cell mediated immunity in mice after cisplatin treatment. Polish. 1 Pharmac Pharmacy 36: 441. 1984. 4 Singh S. Sodhi A: Interaction between cisplatin treated macrophages and Dalton's lymphoma cells in euro. Exp Cell Biol 56: 1. 1989. 5 Muenchen H. Aggarwal S: Immune system activation by cisplatin and its analog ‘poly-plat': an in vitro and in mm study. Anti-CancerDrugs 9: 93. 1998. 6 Palma J. Aggarwal S. Jiwa A: Murine macrophage activation after cisplatin or carboplatin treatment. Anti—cancer Drugs 3: 665. 1992. 7 Adams D. Hamilton T: Activation of macrophages for tumor cell kill: Effector mechanisms and regulation. In: Heppner GH. Fulton AM. eds. Macrophages and Cancer. Boca Raton. FL: CRC Press; 27. 1988. 8 Carleton S. Stevenson A. Hibbs J: Effector mechanisms for macrophage-induced cytostasis and cytolysis of tumor cells. In: Heppner GH. Fulton AM. eds. Macrophages and Cancer. Boca Raton. FL: CRC Press; 39. 1988. 9 Palma J. Aggarwal S: Cisplatin and carboplatin mediated activation of murine peritoneal macrophages in vitro: production of interleukin- la and tumor necrosis factor-a. Anti-Cancer Drugs 6: 1. 1995. 10 Walker E. Gale 0: Methods of reduction of cisplatin nephrotoxicity. Ann Clin Lab Sci II: 397. 1981. ll Fiebig H. Dress M. Ruhnau T. Misra H. Andrulis P. Hendriks H: GB- 21. a novel platinum complex with antitumor activity in human renal and mammary xenografts. Proc Am Ass Cancer Res 37: 297. 1996. 12 Poole A: The detection of lysosomes by vital staining with acridine orange. In: Dingle J. ed. Lysosomes: a laboratory handbook. Amsterdam: ElsevierfNorth Holland Biomedical Press: 313. 1977. 13 Meager A: RIA. IRMA. and ELISA assays for cytokines. In: Ballrwill F. ed. Cytokines: a practical approach. Oxford: Oxford University Press; 299. 1991. 14 Hsu S. Raine L. Fanger H: Use of avidin-biotin-peroxidase Complex (ABS) in immunoperoxidase technique: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29: 577. 1981. 15 Ding A. Nathan C. Stuehr D: Release of reactive nitrogen intermediates from mouse peritoneal macrophages: comparison of activating cytokines and evidences for independent production. J Immunol 141: 2407. 1998. 16 Bucana C. Hoyer L Hobbs B. et al: Morphological evidence for translocation of lysosomal organelles from cytotoxic macrophages into the moplasm of tumor target cells. Cancer Res 36: 4444. 1976. 17 Hibbs J: Heterocytolysis by macrophages activated by Bacrllus Calrnelte Grier-ire lysosome exocytosis into tumor cells. Science 148: 468. 1974. 18 Balkwill F. Naylor M. Malik 5: Tumor necrosis factor as an anticancer agent. Eur J Cancer 2C: 641. 1990. 19 Esumi H. Tannenbaum S: [15.-Japan Cooperative Cancer Research Program: seminar on nitric oxide synthase and carcrnogenesis. Meeting Report Cancer Res 54: 291. 1994. I») 2635 ANTICANCER RESEARCH 18: 2631-2636 (1998) 20 Knowles R. Moncada S: Nitric oxide synthases in mammals. Biochem J 298: 249. 1994. 21 Palma J. Aggarwal S: Cisplatin and carboplatin mediated release of cytolytic factors in murine peritoneal macrophages in vitro. Anti- Cancer Drugs 5: 615. 1994. 22 Hibbs J. Taintor R. Vavrin Z: Macrophage qrtotoxicity: Role for l.- arginine deaminase and amino nitrogen oxidation to nitrite. Serene: 235: 473. 1984. 23 Stuer D. Nathan C: Nitric oxide. A macrophage product responsible for cytoatasis and respiratory inhibition in tumor target cells. J Exp Med 169: 1543. 1989. 24 Krahenbuhl J. Remington J: The role of activated macrophages in specific and nonspecific cytostasis of tumor cells. J Immunol 113: 507. 1974. 33 25 Granger D. Lehninger A- Site of inhibition of mitochondrial electron transport in macrophage-injured neoplastic cells. J Cell Biol 95: 527. 1982 26 Drapier J. Hibbs J: Differentiation of murine macrophages to express nonspecific cytoxieiry for tumor cells results in L-arginine dependent inhibition of mitochondrial iron- sulfur enzymes in the macrophage effector cefls. J Immunol 140: 2829. 1988. 27 anenstein C. Snyder S: Nitric oxide. a novel biologic messenger. Cell 70: 705. 1992. Received February 26. I 998 Accepted April I. I 998 2636 CHAPTER 7: CONCLUSIONS 34 CONCLUSIONS CDDP plus taxol combination stimulates various cytolytic factors of the immune system better than when these drugs are used independently. The macrophage activation includes production of cytoplasmic lysosomes. macrophage/tumor cell contact, and release of cytolytic factors IL-lor and TNF-a. Past in vitro and in vivo studies have shown that when these two drugs are used in combination they are more effective and less toxic than when they are used separately. Our results support activation of the immune system as an additional mechanism of action of this combination therapy. We also propose, based on our observations, that CDDP plus taxol combination activates various cytolytic factors of the immune system better than CDDP or taxol independently. “Poly-plat”, SSP. and SAP induce murine macrophage activation via the production of cytoplasmic extensions. lysosomes. and cytolitic factors IL-la. IL-2 and TNF-a. Previous investigations have shown these drugs to be more effective than cisplatin in vitro and in vivo while eliciting less toxicity. Our studies support the activation of the immune system via the mediation of cytolytic factors and lysosomes as possible mechanisms of action of these drugs. Based on our observations we propose that “poly-plat”. SSP, and SAP activate various cytolytic factors of the immune system bett er, compared to cisplatin. 35 "llllllllllllllllll