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[ RESEARCH 49, 1429-1433, March 15, 1989] Synergistic Antitumor Effects of Topoisomerase Inhibitors and Natural Cell- mediated Teruhiro Utsugi, Sandra Demuth, and Nabil Hanna Department of Immunology, Smith Kline & French Laboratories, King of Prussia, Pennsylvania ¡9406-0939

ABSTRACT elucidated. However, recent studies have demonstrated that topoisomerase II inhibitors, including actinomycin D, synergize The mechanism of augmentation of tumor cell killing by immune with TNF in killing tumor cells sensitive to this cytotoxin effector cells and chemotherapeutic drugs was studied. The effect of (7, 8). treating tumor cells with various antineoplastic drugs on their sensitivity to murine natural cell-mediated Cytotoxicity in vitro was investigated. In the present report, we investigated the potential synergistic Pretreatment with actinomycin D at nontoxic concentrations rendered effect of chemotherapeutic drugs with different mechanisms of L929 and WEHI-164 tumor cells more susceptible to killing by mouse action on natural cell-mediated Cytotoxicity against tumor cells spleen in a dose-dependent manner. Similarly, enhancement and attempted to define the drug and effector cell specificity in of L929 tumor cell killing by natural cell-mediated Cytotoxicity was such a synergy. We demonstrated that treatment with topoi observed following treatment of the target cells with the topoisomerase somerase I or II inhibitors augments the sensitivity of tumor II inhibitors, Adriamycin, , bisantrene, , and tenipo- cells to killing by normal mouse spleen cells. Drugs that act via side, as well as with topoisomerase I inhibitor, . In contrast, mechanisms other than topoisomerase inhibition had weak or drugs which induce their cytotoxic effects by mechanisms that do not no effect. Furthermore, the synergy was most potent with involve topoisomerase inhibition such as , , vincris- effector cells defined previously as NC (9-12) but not with NK tin, and mitomycin C failed to exhibit synergism with natural cell- cells. mediated Cytotoxicity. However, moderate synergy was consistently ob served with m-platinum. The effector cells responsible for the Cytotoxicity in the present system MATERIALS AND METHODS are natural cytotoxic cells since they kill WEHI-164 but not YAC cells, are resistant to treatment with anti-asialo-GMi , and their activity Animals. Specific pathogen-free C3H/HeN mice were obtained from is abolished by anti-tumor necrosis factor . Indeed, tumor Charles River Breeding Laboratories (Kingston, MA). The mice were necrosis factor-mediated Cytotoxicity of WEHI-164 or L929 was en used for experiments at 6-12 weeks of age. hanced by treatment of the target cells with topoisomerase II inhibitors. Drugs. The following chemotherapeutic drugs were used in this study: Moreover, WEHI-164 cells selected for tumor necrosis factor resistance Act D, ADR, BL, CIS, MMC, VB, and VCR were purchased from were resistant to natural cell-mediated Cytotoxicity, and no synergy could Sigma Chemical Co., St. Louis, MO. mAMSA, BIS, CPT, VP16, and be observed with topoisomerase inhibitors. VM26 were kindly provided by Drug Synthesis and Design and Natural Products Branches of the National Cancer Institute, Bethesda, MD. TNF (2 x 10*units/mg ) was provided by J. Chan, Smith Kline INTRODUCTION & French Laboratories, King of Prussia, PA. Target Cells. The following murine tumor cell lines were used: L929 Recent advances in cancer using recombi ; WEHI-164, a chemically induced fibrosarcoma of nant (TNF,1 colony-stimulating factor, 2, BALB/c mice; YAC-1, a lymphoma induced in A strain mice by and ) and immune cells (lymphokine-activated killer Moloney murine virus, and UV-2237, a fibrosarcoma induced cells and T-lymphocytes) render it possible to rationally design in a C3H mouse by chronic UV irradiation. WEHI-164 TNF-R is a TNF-resistant variant of WEHI-164 established by repeated culturing and implement adjunct immunochemotherapy strategies for with TNF-containing medium. L929 and UV-2237 were maintained in cancer treatment. Furthermore, a better understanding of the CMEM at 37°Cin a humidified atmosphere of 5% CO: in air. The molecular mechanisms involved in the destruction of tumor other cell lines were maintained in RPMI 1640. All cell lines were cells by immune effector cells and chemotherapeutic drugs routinely examined for and were found to be free of Mycoplasma would facilitate the selection of drug combinations and proto infection. cols which will result in effective destruction of the tumor cells. Effector Cells. Mice were killed by cervical dislocation and the Synergy between cytotoxic immune cells and antineoplastic spleens were removed. A single cell suspension was prepared by me drugs is well known (1-4). For example, certain tumor cells chanically disrupting the spleens and forcing them through a 60-mesh that are resistant to cytolysis by lymphocytes or wire screen. Erythrocytes in the spleen cell preparations were removed become sensitive to killing following treatment with actinomy by water lysis. In some experiments, nylon wool-nonadherent spleen cin D (1-3). Furthermore, the sensitivity of tumor cells to cells were prepared as described previously (13). Treatment with Chemotherapeutic Drugs. The were cytotoxic products of immune effector cells such as TNF and harvested by treatment with 0.25% trypsin and 0.02% EDTA solution lymphotoxin is enhanced following treatment with actinomycin and were washed once with CMEM. Lymphoma cells grown in suspen D (5, 6). The exact mechanism of this synergy is not fully sion were collected and washed before use. Cells (2.5 x 105/ml) were mixed with various concentrations of drugs in polypropylene tubes and Received 9/30/88; revised 12/5/88; accepted 12/12/88. were incubated for 3 h with occasional shaking in 5% COj at 37"C. At The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in the end of the incubation period, the cells were washed with CMEM to accordance with 18 U.S.C. Section 1734 solely to indicate this fact. remove drugs and then labeled with 5lCr for the Cytotoxicity assay. ' The abbreviations used are: TNF, tumor necrosis factor; NC, natural cyto Antibody-Complement Treatment. Nylon wool-nonadherent spleen toxic; NK, natural killer; Act D, actinomycin D; ADR, Adriamycin; niAMSA. amsacrine; BIS, bisantrene; BL, bleomycin; CIS, m-platinum; CPT, camptothe cells (5 x 106/ml) were treated with anti-asialo-GMi antibody (Wako cin; MMC, mitomycin C; VB, vinblastine; VCR, vincrístine;VP16, etoposide; Pure Chemical Industries, Ltd., Osaka, Japan) at final dilution of 1:50 VM26, ; NCMC, natural cell-mediated Cytotoxicity; CMC, cell-me for 45 min at 4°C.The cells were then washed and incubated with diated Cytotoxicity; anti-asialo GMI, anti-asialo monoganglioside; CMEM, Eagle's rabbit complement at 1:15 dilution (Cedarlane Laboratory, Ltd., minimum essential medium supplemented with 10% fetal bovine serum, sodium Hornby, Ontario, Canada) for 45 min at 37°C.The cells were washed pyruvate, essential amino acids, nonessential amino acids, L-glutamine, 2-fold vitamin solution, penicillin, and streptomycin. and adjusted to 1 x 107/ml before use in the Cytotoxicity assay. 1429 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1989 American Association for . TOPOISOMERASE INHIBITION AND NCMC

100 BL, 0.02-0.1 MM;CIS, 5-25 MM;MMC, 5-25 MM;VB, 1-10 A. MM;and VCR, 2-10 MM. so mAMSA Based on previous studies demonstrating enhancement of -mediated killing by Act D, we examined whether 60 pretreatment with Act D renders L929 fibrosarcoma cells more sensitive to killing by normal mouse spleen cells. L929 cells 40 were incubated with 0-5 MMAct D for 3 h at 37°C;then the cells were washed and labeled with 51Cr before addition to the 20 O VP16 effector cells. This treatment protocol was selected after prelim x ADR inary experiments showed that the drugs do not affect 5lCr O 0.1 1 10 100 0.1 1 10 100 5 [UM] [UM] incorporation or release and that their efficacy is equal to that 100 observed after continued exposure for the 24 h of the cytotox u c. D. icity assay. The results shown in Fig. 2 demonstrate that treat 80 ment with 0.5, 1.0, or 5.0 MMAct D rendered L929 cells more susceptible to CMC as compared to untreated control target cells. Spontaneous 51Cr release from L929 cells treated with 60 CIS 0.1-1.0 MMAct D in the absence of effector cells did not differ 40 BL from controls (26-28% release). A small increase, however, was observed in cells treated with 5.0 MMdrug where spontaneous 20 MMC «VCR release was 6% above control value. The specificity of the synergy with immune effector cells was 0.1 10 100 0.001 0.01 0.1 examined by treating the L929 cells with various chemothera [U/ml] peutic drugs before testing their sensitivity to CMC. Since Act Fig. 1. Direct effects of chemotherapeutic drugs against L929 cells. "Cr- D may inhibit DNA topoisomerase I (15), we evaluated the labeled L929 nbrosarcoma cells were incubated with Act D (A); ADR (A); mAMSA (A); BIS (B); CPT (B); VP16 (B); VM26 (B); CIS (C); MMC (C); effect of topoisomerase II inhibitors (ADR, VP 16), topoisom VCR (C); VB (Q; and BL (D) for 20 h. Percentage of cytotoxicity was determined erase I inhibitors (CPT), and VB which does not act via topoi as described in "Materials and Methods." Data are representative of three separate somerase inhibition. The results shown in Fig. 3 demonstrate experiments. SDs were consistently <10% of the means. that significant enhancement of CMC was observed with to- Cytotoxicity Assay. Mouse spleen cell-mediated cytotoxicity was determined by 51Cr release assay at 4 h for YAC-1, 14 h for WEHI- 60 164, and 18 h for L929 and UV-2237 as described previously (14). Briefly, tumor cells were labeled with 200 ¿iCiof"Cr (Na2CrO4) for 60 È50 min at 37°Cwith occasional shaking and, after washing three times, cells were used as targets. Effector cells were prepared as a single cell 40 suspension as described above. In the assay, a graded number of effector cells were mixed with "Cr-labeled target cells in a total volume of 200 H 30 /il medium per well of 96-well round-bottomed microtiter plates (Cos- tar) and then incubated at 37°Cfor 4 or 18 h in 5% CO: in air. After B2o centrifugaron, 100-jil aliquots of supernatant were harvested and the amount of '''Cr released into the supernatants was measured in a gamma 5? 10 counter. The percentage of killing was calculated according to the O following formula: 6:1 25:1 50:1 100:1

T- S E/T RATIO of cytotoxicity = x 100 M- S Fig. 2. Effect of pretreatment of actinomycin D on mouse spleen cell-mediated cytotoxicity against L929. Act D, 5 MM(A); 1 n\i (•);0.5 ^M (D); 0.1 MM(•);0 (<.)).Data are representative of more than three separate experiments. in which T is cpm released in the presence of effector cells, 5 is cpm released from target cells incubated without effector cells, and M is cpm released from target cells added with 0.1 N NaOH. A. B. Statistical Analysis. The Student t test (2-tailed) was used. All data 60 presented are means of triplicate measurements in representative ex periments out of more than three independent experiments. > 50

= 40 RESULTS 30

To determine the range of drug doses to be used for the 20 synergy experiments with immune cells, we examined the direct effect of drugs against "Cr-labeled L929 cells in a 24-h chro s? 10 mium release assay (Fig. 1). As expected, L929 cells exhibited different degrees of sensitivity to the various chemotherapeutic 6:1 25:1 50:1 100:1 6:1 25:1 50:1 100:1 drugs used. Based on these results, drug concentrations that E/T RATIO E/T RATIO exhibit little or no direct cytotoxicity were used for further Fig. 3. Effect of pretreatment of Adriamycin, etoposide, camptothecin, and studies. These suboptimal concentrations were as follows: Act vinblastine on mouse spleen cell-mediated cytotoxicity. ,•(,mediumcontrol (O); D, 0.1-5 MM;ADR, 5-50 MM;mAMSA, 0.5-5 MM;BIS, 0.5- ADR (D); VP16 (A). B, medium control (•);CPT (•);VB (A). Data are representative of more than three separate experiments. SDs were consistently 2.5 MM;VP16, 5-25 MM;VM26, 0.5-2.5 MM;CPT, 1-10 MM; <10% of the means. 1430

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1989 American Association for Cancer Research. TOPOISOMERASE INHIBITION AND NCMC

poisomerase I and II inhibitors but not with VB. This profile Table 2 Effects of Act D or VP16 pretreatment on susceptibility of murine tumor cell lines to lysis by mouse spleen cells was further confirmed when additional antineoplastic drugs of cytotoxicity"100:1*32.7of with different mechanisms of action were used (Table 1). Treat sponta ment with Act D, ADR, mAMSA, BIS, VP 16, and VM26, all Target neous of which are DNA topoisomerase II inhibitors, enhanced the cellsL929WEH1164YAC-1UV2237DrugMediumrelease21.8 ±2.9C ±1.9 ±0.7 cytotoxicity of spleen cells against L929 by 116, 80, 43, 36,42, Act D(l .«MI 25.1 55.7 ±2.\" 42.4 ±2.3' 29.9 ±1.9* and 42%, respectively. Also, treatment with CRT enhanced the VP 16 (5MM)Medium 21.421.945.5 ±1.4'27.6 43.4±2.1'25.1 36.9 ±1.6'20.3 CMC against L929 cells by 80%. In contrast, drugs which exert ±1.3 51.1 ±4.3"'±1.2 ±3.2 their cytotoxic effect by mechanisms that do not involve topo Act D (0.2 MM) 24.0 64.4 ±4.0* 45.0 ±3.2" isomerase inhibition such as BL, MMC, VB, and VCR had VP16(50MM)MediumAct26.033.042.1 ±2.\"36.3 35.9 ±2.2'33.0 30.3 ±3.021.9 little or no effect on CMC against this target cell. CIS was the ±0.141.0 ±3.2 ±6.1 exception as it consistently enhanced CMC against L929 cells. D(l ,.M) 30.5 ±5.9 38.0 ±1.6 28.0 ±2.3 Having demonstrated that drug specificity exists for synergy VP16(2.5MM)Medium34.829.439.4 ±4.016.4 34.5 ±5.68.4 28.3 ±5.37.1 with normal immune effector mechanisms against one tumor ±2.1 ±0.8 ±3.66.5 target cell, the applicability of this observation to other cell Act D (1 ,AI) 30.9 18.4 ±1.4 11.2 ±2.1 ±4.1 VP 16 (5 MM)% 31.3% 12.3 + 3.250:130.48.2 ±0.325:124.38.3 ±0.7 lines was investigated. This is of importance since different " Percentage of cytotoxicity was assessed by "Cr release assay at 18 h for tumor cells exhibit differential sensitivity to killing by natural L929, 14 h for WEHI-164, 16 h for YAC-1, and 18 h for UV-2237. ' Effectortarget cell ratio. effector cells which include macrophages, NK or NC cells. For ' Mean ±SD of triplicate cultures. Data are representative of more than three this purpose, WEHI-164 fibrosarcoma, UV-2237 fibrosarcoma, independent experiments. and YAC-1 lymphoma cells were used. The direct cytotoxic ' Significantly different from control (P < 0.001). ' Significantly different from control (P < 0.01). effect of the drugs against each target cell was determined and subtoxic concentrations were used for the synergy experiments. The results shown in Table 2 demonstrate that tumor cell effector cells responsible for the synergistic effects observed are treatment with Act D and VP 16 enhanced spleen cell-mediated NC and not NK cells. killing of L929 and WEHI-164 cells but failed to affect the Recently, it was reported that the cytotoxic effects of NC killing of UV-2237 and YAC cells. Considering that the latter cells are mediated through the secretion of TNF (16, 17). Therefore, the potential synergy between TNF and topoisom target cells are killed primarily by NK cells, whereas the former erase II inhibitors in killing L929, WEHI-164, and YAC-1 cells ones exhibit high sensitivity to NC cell killing, it appears that was examined. As shown in Table 4, significant enhancement the synergy between antineoplastic drugs and immune effector of TNF-mediated killing of L929 and WEHI-164 was observed cells is influenced also by the nature of the effector mechanisms following treatment with Act D. Because of the high sensitivity involved in the destruction of the target cells. of WEHI-164 cells to TNF, synergistic effects were maximally To further define the type of effector cells involved in the attained at lower TNF concentrations (10 and 100 units/ml). destruction of L929 and WEHI-164 cells, spleen cells were In contrast, the low levels of TNF-mediated cytotoxicity ob passed over a nylon wool column to remove adherent macro served with YAC cells were not enhanced following treatment phages, then treated with anti-asialo-GMi antibodies and com with Act D. Similar results were obtained by using other topo plement to eliminate NK cells. Such treatment reduced the isomerase II inhibitors (data not shown). CMC against L929 and WEHI-164 cells, while the activity To examine whether the induction of TNF resistance affects against YAC cells was totally abolished (Table 3). Using this the sensitivity to killing by NC cells or the synergy observed effector cell preparation, we were able to demonstrate enhanced with topoisomerase II inhibitors, TNF-resistant WEHI-164 killing of Act D-treated L929 or WEHI-164 cells as compared cells were established by repeated culturing in escalating con to cells not treated with the drug. These data indicate that the centrations of TNF. The results shown in Table 5 indicate that, unlike the parent WEHI-164 cells, the TNF-resistant cells were Table 1 Effects of target cell pretreatment with chemotherapeutic drugs on mouse spleen cell-mediated cytotoxicity against ¿929° completely resistant to killing by mouse spleen cells, a response which could not be affected or enhanced by treatment with 50:1Dose1 of cytotoxicity at Treatment either Act D or VP 16. ofdrugsActinomycin ±0.6* ±4.0(116)""' D MM 54.8 ±1.2(80)"' Adriamycin 25 MM 30.4 ± .9 DISCUSSION mAMSA 2.5 MM 19.4± .9 27.9 ±1.8(43)' Bisantrene 5 MM 30.4 ± .9 41.6± 1.6(36)' VPI6 10 MM 30.4 ± .9 43.4 ±2.0 (42)' In this study we investigated the synergistic activities between 43.2 ±1.7(42)" VM26% 0.5 MM-Drugs27.730.4 ± .9-(-Drugs60.1 antineoplastic drugs and host immune effector mechanisms in 27.7 ±0.6 50.1 ±1.8 (SO)" tumor cell destruction and whether this synergy is restricted to Camptothecin 2 MM a class of drugs with a common mechanism of action and/or to Bleomycin 0.1 unit/ml 30.4 ±1.9 29.6 ±2.5 (-3) 44.5 ±2.6 (60)'' a subpopulation of effector cells. The results demonstrate that c/v Platinum 25 MM 27.7 ±0.6 treatment with DNA topoisomerase I or II inhibitors aug Mitomycin C 25 MM 19.4 ±1.9 25.4 ±4.3 (30) Vinblastine 10 MM 27.7 ±0.6 30.6 ±1.2(10) mented the sensitivity of L929 tumor cells to killing by normal 10 MM 19.4 ±1.9 22.0 ±1.1 (13) spleen cells. Most chemotherapeutic drugs which exert their " The data are representative of more than three separate experiments on each cytotoxic effect by mechanisms other than topoisomerase in drug. Percentages of spontaneous release between control and each drug-treated group were not significantly different. hibition exhibited weak or no effect. The synergy observed with * Mean ±SD of triplicate cultures. m-platinum may be explained by the ability of this drug to c Numbers in parentheses, percentage of enhancement of cytotoxicity by drug produce protein-concealed DNA strand breaks. treatment compared with untreated control. ¿Significantly different from control (P < 0.001). Schlager (4) reported that ADR-treated P815 murine mast- ' Significantly different from control (P < 0.01). cytoma cells were more sensitive to killing by antibody-depend- 1431 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1989 American Association for Cancer Research. TOPOISOMERASE INHIBITION AND NCMC Table 3 Characterization of effector cells mediating cytotoxicity against actinomycin D-treated L929 cells withantibody" ofcytotoxicity*50:125.2 Target cellsDrugL929 100:Ie27.4+1.9'' MediumAct + 1.1 + 1.0 + 20.1 +1.650.9 15.8 ±1.736.6 12.4 ±1.927.3

MM)WEHI D (1 0.2+ ± ±1.0 ±0.3 37.5 ±1.832.1 31.0 ±1.830.6 26.4 ±1.323.3

164 MediumcontrolAct ±0.9 ±1.6 ±2.6 + 20.0 ±1.770.4 17.0±0.665.6 15.6±2.554.5

MM)YAC- D (0.2 ±3.4 ±1.0 ±4.7 + 65.0±1.051.1 43.4+1.540.3 29.6 ±2.929.5

1 Medium controlTreatment ±6.7+ ±7.7 ±4.01.4 2.2 ±3.9% 1.1 ±1.925:120.2 + 2.4 " Antiasialo-GMi antibody was used at final dilution of 1:50 and rabbit complement was followed at 1:15 dilution as described in "Materials and Methods." * Percentage of cytotoxicity assessed at 18 h for L929, 14 h for WEHI-164, and 4 h for YAC-1. Percentages of spontaneous release between control and Act D- treated group on each tumor cell line were not significantly different. ' Effectortarget cell ratio. '' Mean ±SD of triplicate cultures. Data are representative of three independent experiments.

Table 4 Effect of Act D pretreatment on TNF-mediated cytotoxicity against L929, WEHI-164, YAC-1, and UV-2237 tumor cells Act D (units/ml)002.8 of TNF Tumor concentration cells(MM)L929 0 ±0.3 ±0.7 ±0.6 ±0.8 2.5 ±1.2" 4.6 ±1.0 12.3 ±0.3 33.5 ±0.9 48.7 ±1.3 5WEHI 1.9±0.9017.2 6.3 ±0.22.7 15.7±0.823.7 36.8 ±0.754.0 54.4 ±1.168.0

164 0 ±5.4 ±1.6 ±0.4 ±2.5 0.1 ±2.4 55.6 ±2.0 72.6 ±1.3 73.1 ±2.0 73.3 + 2.1 1YAC-1 25.5 ±1.00 54.3 ±2.41.8 67.7 +2.33.5 69.9 ±0.96.4 72.4 ±2.313.9

0 ±3.2 ±3.4 ±2.6 ±2.4 1 0 0 0 02.4 14.9 ±1.9 5UV-2237 00 00 00 ±2.10.1 15.1±2.82.6

0 ±0.118.4 ±3.823.7 1 10.1 ±3.9 10.9 ±1.4 10.7 ±0.6 ±0.7 ±2.0 5Concentration 11.4 ±2.0100.3 9.8 ±1.01003.3 14.3 ±4.31,00016.1 17.7 ±1.210,00028.9 23.2 ±2.6 " Percentage of cytotoxicity assessed at 20 h for each tumor cell. Mean + SD of triplicate culture. Data are representative of three independent experiments.

Table 5 Effects of target cell pretreatment of Act D or VPÌ6onmouse spleen cell-mediated cytotoxicity against WEHI-164 and WEHI-164-TNF-resistant cells ofcytotoxicity"50:127.2 Target spontaneous cellsWEHI release20.3 164WEHI164TNF-RrDrugsMedium ±3.8C ±2.2 ±2.0 Act D (0.1 MM) 20.8 51.0+ I.!' 44.9 ±2.1'' 37.8 ±0.6'' VP16(50MM)Medium 24.126.8 45.4 ±2.0*5.2 40.8 ±lV1.8 35.2 ±2.1'0

±0.6 + 0.3 Act D (0.1 MM) 34.2 8.6 ±1.7 3.1 ±2.0 2.1 ±1.6 VP 16 (50 MM)%of 31.9100:1*30.1 8.8 ±1.4% 3.8 ±2.025:123.6 2.8 + 0.8 " Percentage of cytotoxicity assessed at 14 h. * Effectortarget cell ratio. ' Mean ±SD of triplicate cultures. d Significantly different from control (P < 0.001). ' Significantly different from control (P < 0.01). F-R, TNF-resistant cells. ent cytotoxicity and MMC-treated P815 cells showed enhanced mor target cells by topoisomerase inhibitors. The results ob sensitivity to killing by cytotoxic T-cells. In this study, the tained with Act D, ADR, and VCR are consistent with those mechanism of augmentation apparently involved a change in of Coletta et al. (18). It should be emphasized, however, that the physical properties of the . Other investiga this observation does not apply to all tumor cells evaluated nor tors (1-3, 18) have demonstrated that preincubation with Act to all effector immune mechanisms. For example, UV-2237 D induced selective sensitivity of WEHI-164 to - fibrosarcoma and YAC-1 lymphoma cells failed to show en mediated killing. Additionally, of the different chemotherapeu- hanced sensitivity to NCMC after treatment with topoisomer ase inhibitors. In contrast, WEHI-164 cells behaved in a fashion tic drugs tested, ADR and cyclohexamide had an enhancing similar to that of L929 cells. This difference, however, may be effect; whereas VCR, 5-, , and 1,3- dependent on the nature of the effector cells involved in tumor bis(2-chloroethyl)-l-nitroso-urea were ineffective (18). In this cell killing, as WEHI-164 and L929 are killed primarily by study, using different tumor target and effector cells, we dem natural cytotoxic cells (16, 17) (Table 2); whereas YAC-1 and onstrated that the sensitivity to killing by normal spleen lym UV-2237 tumor cells are sensitive to NK cell-mediated cyto phocytes is enhanced primarily following treatment of the tu toxicity (16, 19). 1432 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1989 American Association for Cancer Research. TOPOISOMERASE INHIBITION AND NCMC

Natural cytotoxic cells are present in mouse spleens and mycin D and etoposide was obtained in vivo following combi human peripheral blood and exhibit spontaneous cytotoxicity nation with TNF (8). similar to that described for NK cells (9). The two cells, how Together, our data and previously published reports may ever, differ by surface antigenic markers, kinetics of tumor cell provide a basis for a rational selection of chemotherapeutic lysis, and tumor cell specificity (9-11). Moreover, the mecha drugs and immunomodulators that when used in combination nism of NC- but not NK-mediated killing involves the secretion therapy would synergize for tumor destruction in vivo. of TNF (16, 17). Indeed, treatment with anti-TNF antibodies abolished NCMC against WEHI-164 and L929 and no synergy REFERENCES with Act D could be observed.2 In contrast, treatment with anti- asialo-GMi only marginally reduced NCMC and failed to affect 1. Ziegler-Heitbrock, H. W. I... and Riethmuller, G. A rapid assay for cytotox icity of unstimulated human . J. Nati. Cancer Inst., 72: 23-29, the synergy with Act D. 1984. The possibility that the synergistic activity between NC cells 2. Coletta, F., Peri, G., Villa, A., and Mantovani, A. Rapid killing of aitino mycin D-treated tumor cells by human mononuclear cells. I. Effectors belong and topoisomerase inhibitors is mediated by enhanced tumor to the monocyte-macrophage lineage. J. Immunol., 132:936-944, 1984. target cell sensitivity to TNF-mediated cytotoxicity is supported 3. Coletta, F., Bersani, L., Lazzarin, A., Poli, G., and Mantovani, A. Rapid killing of actinomycin D-treated tumor cells by human monocytes. II. Cyto by the following evidence, (a) Induction of TNF resistance toxicity is independent of secretion of reactive oxygen intermediates and is renders WEHI-164 cells resistant to NC-mediated killing and suppressed by protease inhibitors. J. Immunol., 134: 3524-3531, 1985. to synergy with chemotherapeutic drugs; (b) synergy was ob 4. Schlager, S. I. Ability of tumor cells to resist humoral IM.cell-mediated immune attack in controlled by different membrane physical properties. served between Act D and TNF in killing L929 and WEHI-164 Biochem. Biophys. Res. Commun., 106: 58-64, 1982. cells but not YAC-1 and UV-2237 cells which are relatively 5. Flick, D. A., and Gifford, G. E. Comparison of in vitro cell cytotoxic assays resistant to TNF-mediated cytotoxicity; and (c) drug specificity for tumor necrosis factor. J. Immunol. Methods, 68: 167-175, 1984. 6. Kornbluth, R. S., and Edgington, T. S. Tumor necrosis factor production by for synergy with TNF has been demonstrated (7, 8) to be similar human monocytes is a regulated event: induction of TNF-a-mediated cellular cytotoxicity by endotoxin. J. Immunol., 137: 2585-2591, 1986. to that described for NC cells in the present study. 7. Alexander, R. B., Nelson, W. G., and Coffey, D. S. Synergistic enhancement The mechanisms by which topoisomerase inhibitors render by tumor necrosis factor of in vitro cytotoxicity from chemotherapeutic drugs tumor cells susceptible to immune effector mechanisms, such targeted at DNA topoisomerase II. Cancer Res., 47: 2403-2406, 1987. 8. Alexander, R. B., Isaacs, J. T., and Coffey, D. S. Tumor necrosis factor as NC cells and TNF, have not been fully elucidated. The enhances the in vitro and in vivo efficacy of chemotherapeutic drugs targeted effects, however, are most likely related to an increase in the at DNA topoisomerase II in the treatment of murine . J. I rol., level of topoisomerase-mediated DNA damage. Recent studies 138:427-429, 1987. 9. Stutman, ( ).. Paige, C. J., and Figarella, E. F. Natural cytotoxic cells against have demonstrated that DNA fragmentation is induced by TNF, solid tumors in mice. I. Strain and age distribution and target cell suscepti lymphotoxin, and cytotoxic T-lymphocytes, and that DNA bility. J. Immunol., 121:1819-1826, 1978. 10. Paige, C. J., Figarella, E. F., Cuttito, M. J., Cahan, A., and Stutman, O. damage is a key process in the cytotoxicity exhibited by these Natural cytotoxic cells against solid tumors in mice. II. Some characteristics of the effector cells. J. Immunol., 121: 1827-1835, 1978. immune effector mechanisms (20, 21). Therefore, it would not 11. Liuinu-, E. I,., Pecoraro, G. A., and Stutman, O. Natural cytotoxic cells be surprising to find synergy between topoisomerase inhibitors against solid tumors in mice. III. A comparison of effector cell antigenic and NC cells or TNF since DNA cleavage, albeit induced via phenotype and target cell recognition structure with those of NK cells. J. Immunol., 126: 2011-2014, 1981. different molecular mechanisms, constitutes, a central event 12. Djeu, J. Y., Lanza, E., Pastore, S., and Hapel, A. J. Selective growth of leading to cell death in both cases. This is further supported by natural cytotoxic but not natural killer effector cells in interleukin-3. Nature the observation that synergy with topoisomerase inhibitors was (Lond.), 306: 788-791,1983. 13. Julius, M. H , Simpson, E., and Herzenberg, L. A. A rapid method for the not observed with tumor cells selected for resistance to killing isolation of functional thymus derived murine lymphocytes. Eur. J. Immu by NC cells or TNF. The possibility of a direct drug-mediated nol., 3:645-650, 1973. 14. Hanna, N. Inhibition of experimental tumor by selective activation activation of the effector cells (22) could be ruled out as being of natural killer cells. Cancer Res., 42: 1337-1342, 1982. responsible for the enhanced cytotoxicity, since pretreatment 15. Trask, D. K., and Muller, M. T. Stabilization of -DNA covalent complexes by actinomycin D. Proc. Nati. Acad. Sci. USA, 85:1417- of the target cells with topoisomerase inhibitors followed by 1421, 1988. extensive washing was as effective as maintaining the drugs 16. Ortaldo, J. R., Mason, L. H., Mathieson, B. J., Liang, S-M., Flick, D. A., and Herberman, R. B. Mediation of mouse natural cytotoxic activity by throughout the incubation with the effector cells. This obser tumor necrosis factor. Nature (Lond.), 321: 700-702, 1986. vation, together with results from experiments measuring cel 17. Patek, P., Lin, Y., and Collins, J. L. Natural cytotoxic cells and tumor lular uptake of radiolabeled drug,2 rule out the possibility that necrosis factor activate similar lytic mechanisms. J. Immunol., 138: 1641- 1646, 1987. enhanced drug uptake by the tumor target cells is responsible 18. Coletta, F., Polentanitti, N., Bersani, L., Poli, G., and Mantovani, A. Rapid for the enhanced tumor cell destruction observed in the present killing of actinomycin D-treated tumor cells by mononuclear phagocytes: characterization of effector cells in mice. J. Leukocyte Biol., 39: 205-221, system. 1986. The clinical relevance of the synergy between selected chem 19. Hanna, N. -mediated cytotoxicity against solid tumors: in vitro and in vivo studies. In: R. B. Herberman (ed.), NK Cells and Other otherapeutic drugs and host defense mechanisms remains to a Natural Effector Cells, pp. 1359-1367. New York: Academic Press, 1982. large extent unknown. In experimental models, however, evi 20. Schmid, D. S., Tite, J. P., and Ruddle, N. H. DNA fragmentation: manifes tation of target cell destruction mediated by cytotoxic lines, lympho- dence has been provided in support of synergistic antitumor toxin secreting helper T-cell clones, and cell-free lymphotoxin-containing effects between chemotherapeutic drugs and host immunity supernatant. Proc. Nati. Acad. Sci. USA, 83: 1881-1885, 1986. (21). Similarly, using murine bladder, tumor enhancement of 21. Dealtry, G. B., Naylor, M. S., Fiers, W., and Balkwill, F. R. DNA fragmen tation and cytotoxicity caused by tumor necrosis factor is enhanced by the antitumor efficacy of the topoisomerase inhibitors actino- interferon-7. Eur. J. Immunol., 17:689-693, 1987. 22. Mantovani, A. The interaction of cancer agents with mono- 2Unpublished data. nuclear phagocytes. Adv. Pharmacol. Chemother., 19: 35, 1982.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1989 American Association for Cancer Research. Synergistic Antitumor Effects of Topoisomerase Inhibitors and Natural Cell-mediated Cytotoxicity

Teruhiro Utsugi, Sandra Demuth and Nabil Hanna

Cancer Res 1989;49:1429-1433.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1989 American Association for Cancer Research.