Effects of Cytochalasin B in Culture and in Yivo on Murine Madison 109 Lung Carcinoma and on B16 Melanoma1

ICANCER RESEARCH 50. 1431-1439. March 1. 1990] Effects of Cytochalasin B in Culture and in yivo on Murine Madison 109 Lung Carcinoma and on B16 Melanoma1

Peter F. Bousquet, Lesa A. Paulsen, Christopher Fondy, Karen M. Lipski, Karen J. Loucy, and Thomas P. Fondy2

Department of Biology, Syracuse University, Syracuse, New York 13244

ABSTRACT neoplastic cells (6-26). Effects on cell motility, adherence, secretion, and drug efflux, among other effects, suggest to us Cytochalasin B (CB), at 100 or at 10 mg/kg single dose s.c. in carboxymethyl-cellulose (2%)/Tween-20 (1%) 24 h after s.c. challenge that the cytochalasins might produce significant responses in of 1561)21i mice with trocar implants of B16F10 tumor s.c., delayed the experimental cancer chemotherapy model systems in vivo either appearance of measurable tumor nodules by 157 and 93%, respectively, as individual agents or, more likely, as agents amplifying re and extended host survival by 65 and 26%. Tumor growth was also sponses produced by known antitumor drugs. delayed when CB treatment was given 1 day after the appearance of For reasons detailed earlier (2), very few in vivo studies have palpable tumor nodules. By in vivo bioassay, in vitro cloning, and dye been carried out with the cytochalasins. In vivo toxicities of exclusion measurements, solid tumor nodules treated in vivo with CB at various cytochalasins, Cytochalasin derivatives, and chaetoglo- either 100 or 10 mg/kg showed the same viability and tumorigenicity as bosins (27-29) have been investigated, but it does not appear did vehicle-treated nodules 4 and 6 days after drug treatment, at which that CB3 itself has ever been evaluated in mice for in vivo time growth inhibition was still apparent. This indicates that growth pharmacodynamic effects of any kind. Some direct attempts at inhibition by CB is not dependent on a gross cytotoxic effect. (1)21, cancer chemotherapy against LI210 leukemia. Ehrlich carci mice challenged s.c. with Madison 109 lung carcinoma cells and treated with CB s.c. at 100 or 150 mg/kg 24 h later showed a 66% delay in the noma, sarcoma 180, and some other neoplastic model systems in rodents have been made using Cytochalasin D (27-30). How median day of tumor nodule appearance. When administered under these conditions or at the time of nodule appearance, CB markedly inhibited ever, these were tests designed to evaluate single-agent cytotoxic the rate of tumor growth, prevented tumor invasion at day 23, extended antitumor activity, a property unlikely to be important in cy life span by 23%, and significantly inhibited spontaneous lung mÃ©tastases tochalasins, which are not especially cytotoxic. These early measured 28 days after tumor challenge. Maximum tolerated doses of chemotherapeutic studies did not reveal direct cytotoxic anti- CB administered i.p., s.c., or i.v. in suspension or in solution are defined. tumor activity in vivo. These results delineate the conditions under which CB can be tested for In experimental designs more relevant to the unusual prop in vivo biological activities and establish that this microfilament-active natural product in a single-agent protocol inhibits local tumor growth erties of the cytochalasins, B16F10 murine melanoma cells (31) and extends survival in B16F10 melanoma and Madison 109 lung carci and TA3 murine mammary ascites carcinoma cells (32) were noma, and, in the latter model, inhibits invasion and spontaneous lung treated with CB in vitro and transplanted in vivo, but, unfortu mÃ©tastasesbymechanisms that do not appear to depend on cytotoxicity. nately, without the effect-sustaining direct administration of This work on formulation, tolerated doses in vivo, and localized peritu- CB in vivo. Intravenous challenge with B16F10 cells treated moral effects of CB now permits evaluation of systemic antitumor effects with CB under these conditions showed increased extrapulmo- of Cytochalasin B as a single agent. It also permits chemotherapy studies nary mÃ©tastases(31). However, many of the important mor using CB as a potential amplifier of the activity of other antitumor agents phological and functional effects of cytochalasins in vitro are known to be readily reversible (references detailed in Ref. 2). Thus, in vitro alteration of cells with cytochalasins followed by INTRODUCTION implantation in vivo without the additional administration of cytochalasins in vivo is not likely to reveal the full potential of Cytochalasins form a congeneric family of more than 24 Cytochalasin effects on cell functions in vivo. structurally related secondary metabolites produced by various A full evaluation of the in vivo utility of these agents will species of molds (1). These agents exert profound effects at require that bioactive concentrations be achieved in target tis micromolar and submicromolar concentrations on the mor sues and maintained for times sufficient to produce pharma phology and functions of a wide variety of cells and tissues in codynamic effects. Such in vivo evaluation necessarily requires culture [for a detailed review see the preceding paper in this that the agents be available in amounts that are generally series (2) and two comprehensive review books (3, 4)]. The impractical to obtain from commercial sources. It also requires morphological and functional effects produced by cytochalasins knowledge concerning formulation, administration, and dose- appear to arise largely because of alteration in actin polymeri zation, in turn affecting the structure, function, and plasma limiting host toxicities as a function of formulation, route of membrane interactions of microfilaments (5). Thus the cyto administration, and dose scheduling. Moreover, evaluation of chalasins are considered to be microfilament-directed agents in in vivo interactions of cytochalasins with other antitumor agents the same sense that the Vinca alkaloids are microtubule di necessarily requires that the effects of the cytochalasins as single rected. agents be known. We have developed improved procedures for A very large number of studies have been carried out on the the production of CB and have determined its distribution as a effects of cytochalasins on cell and tissue morphology and function of time in 14 tissues following single dose bolus i.p. function in vitro, including comparisons among normal and injection (2). In the present work we have determined the maximum tolerated doses of CB administrated by different Received 2/15/89; revised 7/21/89. 11/13/89; accepted 11/27/89. routes as a bolus drug in a variety of formulations. The earlier 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 CB production and tissue distribution work along with the accordance with 18 U.S.C. Section 1734 solely to indicate this fact. toxicity studies presented here permit us to examine the anti- ' Supported by the Stella Hardeman Memorial Grant for Cancer Research to the American Cancer Society and by the Liposome Company. Princeton. New 3The abbreviations used are: CB, Cytochalasin B: CM-cellulose. carboxyme- Jersey. thycellulose; M109. Madison 109; DMSO. dimethyl sulfoxide; HPLC. high 1To whom inquiries should be addressed. performance liquid chromatography; TLC, thin layer chromatography. 1431

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1990 American Association for Cancer Research. CYTOCHALASIN B AND B16 AND M109 TUMORS /A' VIVO tumor effects of cytochalasin B as a single agent in vivo in Cytochalasin B. The compound was prepared in lots of 400 to 600 anticipation of later studies with in vivo drug combinations. mg from mold mattes of Drechslern dematioidea by the method of The present work details our results in culture, in vivo with Lipski et al. (2), was purified by preparative TLC, and was analytically the B16F10 melanoma syngeneic in C57BL/6 mice, and in vivo pure at the 99% level as detailed in that work. Radioactive CB was with the Madison 109 murine lung carcinoma syngeneic in prepared as described previously (2). In Vitro Growth Curves. B16F10 cells were routinely harvested from BALB/c hosts, a carcinoma which is invasive and metastatic flasks at late log phase, seeded into 24-well culture plates (Corning) at from a s.c. implant. Using these systems we have determined a concentration of 2.5 x 10' trypan blue excluding cells/cnr into wells effects on growth kinetics, localized invasion, spontaneous mÃ© containing 2.0 ml complete medium, and allowed to attach overnight. tastases, and host survival. After 24 h the medium was removed and replaced with 2.0 ml of fresh complete medium. The wells were treated with various concentrations of CB dissolved in DMSO. CB solutions were prepared from a stock MATERIALS AND METHODS solution of 1 mg/ml, which was stored frozen and protected from light. CB was added in 5- to 20-^1 aliquots and all growth experiments Animals and Tumors. The M109 lung carcinoma arose spontaneously in 1964 in the lung of an 18-month-old BALB/c mouse. The tumor involved control wells, which received the equal volume of DMSO (0.25% to 1% final DMSO concentration). Identical wells were har was characterized as an alveologenic carcinoma and has been highly vested in triplicate or quadruplicate, counted in a Coulter counter, and consistent in its histology and growth characteristics (33, 34). Its value averaged to determine absolute cell number for each CB concentration. as a chemotherapeutic model system has been examined using virtually Viability was determined by trypan blue exclusion. every known clinically active antineoplastic agent in a thorough analysis Reversibility in Vitro. Cells were grown and monitored as in growth by Rose (35). The M109 lung carcinoma used in this work was obtained curve experiments but were washed at various times. Washing involved from Dr. William Rose (Bristol Laboratories). All of our work with removal of medium, addition of 2.0 ml fresh complete medium, gentle M109 was conducted with BALB/c and CD2F, (BALB/c female x pipetting, removal of wash medium, and addition of fresh medium. DBA/2 male) mice. The M109 carcinoma was maintained by serial s.c. passage of 20% tumor brei (approximately 1 x IO6total cells) in BALB/ This procedure was repeated two additional times. c mice every 12 days (35). Experimentation was done on CD2F, mice Determination of CB in Treated Cells and Media. Plates were seeded and treated as in growth curve studies. For each concentration three with M109 implanted either i.p., i.v. through the tail vein, or s.c. wells were harvested and pooled at the indicated times. Medium with ventrally on the abdomen. Tumor brei for passage was prepared by excising the tumor from the residual CB was separated from cells and each was analyzed separately. passage mouse, removing any necrotic areas, weighing the tumor, For analysis of CB in the medium. 5 ml of HPLC grade methanol were mincing it aseptically, and suspending the fragments in 0.85% (w/v) added to the 6 ml of pooled medium and centrifuged at 1000 x g for 5 sterile NaCI solution (l g tumor/ml) containing 0.4 units of penicillin min and the supernatant was collected. HPLC grade water (15 ml) was and 0.4 n% of streptomycin/ml. The suspension was then diluted to added to give a 20% methanol final concentration. This was then passed 20% with 0.85% sterile NaCI solution and 0.2 ml (2 x IO5trypan blue- through a CIS Sep-Pak (Waters) that had been washed with 4 ml excluding cells) was implanted s.c. ventrally on the abdomen. For drug methanol followed by 6 ml HPLC grade water. testing, the 20% cell suspension was diluted to allow injection of 1 x For analysis of CB in cells, the cells were harvested as in growth 10' trypan blue-excluding cells per mouse. Trypan blue exclusion was experiments and 5 ml methanol were added. This mixture was homog determined using 0.2% dye solution in a 1:1 (v/v) ratio with cell enized with a Polytron and centrifuged and the supernatant was col suspension. Viability of the tumor suspension by dye exclusion was lected. This solution was made 20% methanol by adding water and passed through a Sep-Pak cartridge as above. Extracts from cells or generally around 20%. B16F10 murine melanoma selected by Fidler (36) for high lung- media were treated identically after this point. After drying overnight colonizing potential from i.v. challenge was received from the Division in a vacuum desicator, the samples were eluted from the CIS cartridge of Cancer Treatment, Tumor Depository. This subline was maintained with 2 ml HPLC grade ethyl acetate. The eluates were dried down in culture in RPM1 1640 medium, containing 20 ITIM4-(2-hydroxy- under nitrogen and redissolved in 70 ^1methanol and 50 Â¿ilw-ereloaded ethyl)-l-piperazineethanesulfonic acid buffer, 100 ng/ml streptomycin, onto an analytical Linear K silica gel TLC plate with a sample- 100 units/ml penicillin, 250 Mg/ml fungizone, 10% newborn calf serum concentrating pre-run zone. The plate was run in a solution of 15% (Grand Island Biological Company), and 2 g/liter sodium bicarbonate methanol/85% toluene (v/v) or 3/1 benzene/acetone, dried, and stained (hereafter called complete medium), and 5% carbon dioxide at 37Â°C with p-anisaldehyde as detailed previously (2). CB (1 and 5 ^g) was (pH 7.2). All media were filter-sterilized immediately prior to use. Cells added to parallel cell and media samples as controls and to TLC plates were harvested in log phase by detaching with 0.25% trypsin/0.02% directly as a marker. EDTA for 1 min or less and then adding 10 ml of complete medium. In Vivo Chemotherapy Testing. When testing was done on animals The line was maintained by subculturing late log phase cells by 1:10 challenged with either B16F10 or M109 tumors passaged in vivo, the dilution every 5-6 days. The original cell sample from the National animals were challenged either with a cell suspension (1 x 10' trypan blue-excluding cells for B16F10 or 1 x IO5trypan blue-excluding cells Cancer Institute was expanded and frozen in complete medium brought up to 50% fetal bovine serum, 10% DMSO, v/v. After 10 passages, for M109) s.c. or Â¡.p.,ors.c. with an implant of tumor pieces (0.5-mm new stock was thawed, washed in complete medium, and maintained cubes) with a 13-gauge trocar on day 0. Tumor treatment was initiated in vitro. B16BL/6, a subline selected for bladder connective tissue either on day 1 or at the time of appearance of palpable tumor nodule. invasiveness (37), was also obtained from the Division of Cancer Animals were sedated for trocar implant with Nembutal at 40 mg/kg. Treatment, Tumor Depository, and maintained as above for the FIO One day prior to tumor challenge, hair in the region of trocar entry subline. and at the site of tumor deposition was removed with electric clippers The FIO and BL6 sublines were used as challenge tumors either as and chemical depilatory to permit sterile sealing of the entry wound suspensions prepared from cultured cells, or as trocar implants of solid and to facilitate caliper measurement of tumor dimensions. tumor pieces prepared from s.c. tumors elicited by injection of sus CB Treatment. The CB was administered in solution in DMSO (s.c.) pended cultured tumor cells. Subcutaneous trocar implantation of a or suspended in 0.85% sterile NaCI solution (i.p. and s.c.) or in sterile solid piece of tumor (0.5 mm') on the ventral surface of C57/BL6 mice pyrogen-free 2% CM-cellulose/1% Tween-20 (s.c., Â¡.p.,or i.v.) (a gift was performed under Nembutal anesthesia with a 13-gauge tumor from Dr. Thomas Tice, Southern Research Institute. The CM-cellu- implant needle (trocar). In experiments where in vivobioassay of treated lose/Tween was found to be the most reliable vehicle for administration tumors was performed, single-cell suspensions were prepared by asep of CB in suspension, since this vehicle minimized adherence of CB to tically removing the treated tumors, mincing in sterile Â¡sotonicNaCI the syringe walls and permitted delivery of 80 to 90% of the nominal solution, and passing the suspension through a sterile nylon filter to dose, as determined by TLC analysis of the residual CB in the syringes. remove cell clumps and connective tissue. For i.v. administration of CB in solution, the CB was dissolved in 1432

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1990 American Association for Cancer Research. CYTOCHALASIN B AND B16 AND MI09 TUMORS IN VÃŒVO ethanol and diluted to 48% ethanol using warm pyrogen-free NaCl solution immediately prior to tail-vein injection (0.1 ml total vehicle). Dilution to 33% ethanol also retained CB in solution but was not used in this work. Suspensions were achieved by weighing CB into a syringe, loading the vehicle into a second syringe, connecting the syringes with a double- hubbed emulsifying needle, forcing the suspension back and forth repeatedly through the coupling tube until flow was impeded, and then progressively decreasing the bore diameter from 21 gauge to 23 gauge and finally to 25 gauge. Syringes and couplers were washed with methanol and the methanol wash was analyzed by TLC to determine residual CB that was not actually delivered to the test animals. CB Toxicity in Vivo. Maximum tolerated single bolus doses (LD5_10) as a function of vehicle and route of administration were estimated by cumulative experience with CB treatment of tumor-bearing animals CB (either M109 in CD2F or B16 melanoma in B6D2F, mice). Maximum tolerated doses estimated in tumor-bearing animals were confirmed in Fig. 1. Growth of B16F10 melanoma cells in vitro in the continuous presence of cylochalasin B. Cells were seeded at 2.5 x IO4cells/cm2 at time 0. allowed 10 non-tumor-bearing CD2F,, B6D2F,, or C57B1/6 mice. 20 to 24 g, of grow for 24 h. and then treated with CB in DMSO. DMSO concentrations ranged either sex. The effects of subacute CB doses were monitored by deter from 0.25 to 1.0% and did not affect cell proliferation significantly. Cell growth mination of animal weights daily for 2 weeks. The vehicles employed was determined by Coulter counter after eell detachment. [CM-cellulose/Tween or 1:1 (v/v) 95% ethanol/0.85% NaCl solution] were pyrogen free (Limulus amebocyte lysate assay; Whittaker M. A. and 4 days. A sharp cytostatic dose-response effect was ob Bioproducts, Walkersville, MD). Effects on Primary Tumor and MÃ©tastases.Primary s.c. tumor grow th served, where cells treated by continuous exposure to 200 n\i was monitored by noting the day when tumor first became measurable. CB grew to 70% of the density reached by DMSO-treated Growth rate was monitored by calculating tumor weight, using controls after 4 days, while cells treated at l /IM grew to only measurement of two perpendicular dimensions and the equation 10% of the control cell density. CB at 4 /Â¿Mwascompletely (L x w2)/2 (38), and by host survival where animals were terminated cytostatic, but no evidence of cell destruction was seen even when tumors reached 25 mm in one dimension or when an animal after 4 days of drug exposure in vitro. An IC5n value of 460 n\i appeared to be in distress. The validity of the tumor growth rate was determined after 3-day exposure and 370 HMafter 4 days. measurements was tested by comparing actual weights of tumors ob A departure from linearity was observed at the 1CÂ«?pointwhen tained at autopsy to values obtained prior to the terminal stages. exposure to CB was for 3 days. This does not affect comparisons Intraperitoneal and i.v. tumor growth was determined by host survival, of ICso values but does suggest a concentration-dependent self- which included animals terminated at the point where distress was apparent and end stages predictable within 2-3 days. Invasion from s.c. limiting effect, possibly cell cycle based, and has been observed by us with other neoplastic cell lines.4 implants of M109 tumor was readily apparent as breaking through the abdominal wall and into the peritoneum. MÃ©tastasesinthe liver, spleen, Final DMSO concentrations in vitro ranged from 0.25 to lungs, and pancreas were determined either by necropsy of terminal 1.0% and produced a maximum inhibition compared to un stage animals or by necropsy of animals sacrificed at a constant time treated control cells of less than 30%. Cells exposed to CB at point, generally 23 to 30 days after tumor implant, as indicated. Organs were fixed in Bouin's solution (75 ml saturated picric acid, 25 ml levels above 100 nM showed alterations in morphology. The cells became rounded, rather than exhibiting the fibroblastic formalin, and 5 ml glacial acetic acid) and metastatic foci were counted spreading characteristic of DMSO-treated control cells or of under a dissecting microscope. MÃ©tastasesare presented as the propor untreated cells, and the CB-treated cells had additional pseu- tion of animals with one or more metastatic nodules and also as the mean number of nodules per organ. dopodal appendages. These morphological changes appeared Measuring Viability of Tumor Cells in Vivo. Tumor cell viability was to be directly proportional to both the concentration and the determined by three methods: trypan blue exclusion, plating efficiency, time of exposure (data not shown). and growth of tumor in secondary' recipients. In all cases, s.c. tumors Reversibility of the Cytostatic Effect in Vitro. According to a were removed from initial experimental mice, minced, and dissociated number of studies (cited in Ref. 2), most of the effects of CB to form a single-cell suspension in sterile isotonic saline. A 0.1-ml on cells are rapidly reversible upon removal of the CB-contain- aliquot of suspension was added to 0.1 ml of 2% trypan blue. Total ing growth medium followed by washing and refeeding of the cells and viable cell counts were made on a hemacytometer. Secondary isogenic recipients were inoculated with 1 x IO6 viable cells s.c. and cells with fresh medium. In order to test the reversibility of the cytostatic effects produced by CB, cells were exposed to 1.0 or monitored for tumor appearance and host survival. 5.0 tiM CB for 7, 24, or 48 h prior to removal of the CB- To determine plating efficiency, cells were plated in 6-well tissue culture plates (Costar) at a density of 1 x 10' cells/cm2. Cell colonies containing medium, washing, and refeeding with fresh medium (50 or more cells clustered) were counted on day 4 of culture. Plating (Fig. 2). After a 24-h period to allow attachment and equilibra efficiency was defined as the ratio of the number of colonies to the tion of the washed cells, growth rates for the various cell number of cells plated, expressed as percentage. treatment groups were determined. The reversibility of the Statistics. Significance was determined by Student's t test and by effects of CB in this system was a function both of CB concen Wilcoxon-Mann-Whitney two-sample test. tration and of duration of exposure to CB. Cells exposed to 1.0 Â¿IMCB(Fig. 2/1) for either 7 or 24 h and then washed grew within 3 days after CB removal to a 4-fold greater density than RESULTS cells continuously exposed to 1.0 /KMCB (unwashed controls). Under the same conditions, the cells which were exposed to 1.0 Effect of CB on B16F10 Growth and Morphology in Vitro. n\i CB for 48 h prior to removal of CB took 6 days for the B16F10 cells were treated in vitro with concentrations of CB washed cells to achieve a 4-fold increase in density compared ranging from 100 n\i to 4 UM 24 h after seeding in vitro, at which time the cells had a density of 3 x lO^/cm2. Fig. 1 shows to cells continuously exposed to CB at l UM. A drug exposure the dose-response effects of continuous exposure to CB for 3 4J. M. Mitchell and T. P. Fondy. unpublished observations. 1433

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1990 American Association for Cancer Research. CYTOCHALASIN B AND B16 AND M109 TUMORS IN VIVO Table 1 Cytochalasin B: bolus dose-limiting acute toxicities in mice 400 CB treatment deaths (mg/kg/day)0CB fraction2/34c'" administered i.p. 50 50' CMC/Tw 0/24C'" 10025 CMC/TwDMSO 5/9f'"'Â£6/jf0/5*

50CB DMSOCMC/Tw

administered i.v. 10 20 CMC/Tw 0/5" 40 CMC/Tw 2/2" 50510 CMC/TwEtOH/NaCI' 2/2"0/29*

EtOH/NaCl 0/4f 2010 EtOH/NaClEtOH/NaCl 3/4f0/5"

0/7" 2 3456 20 EtOH/NaCl Days Post Washing 40CB EtOH/NaClCMC/Tw 2/2"0/31'-" Fig. 2. Reversibility of CB effects in vitro. B16F10 cells were grown and administered s.c.' treated as for Fig. 1 but were rendered drug-free by repeated washing with fresh 100 medium at the times indicated. Cell growth was determined daily by Coulter ISO'100 CMC/TwDMSO 4/28c5/1 counter and compared to washed and unwashed control wells and to unwashed drug-treated wells. A, 1 pM CB; B, 5 H.MCB. 6C 100* DMSO 0/1 Ã³" 150VehicleCMC/Tw* DMSODrug 6/10' of only 7 h at 5.0 ^M CB (Fig. 2B) was sufficient to produce " Day 1 only unless noted otherwise. the same level of growth inhibition as was produced by 1.0 //M * CM-cellulose, 2%/Tween-20, 1%, tested pyrogen-free. CB after 48 h of exposure. When cells were treated with 5.0 CCD2F|. normal or M109 tumor-bearing. " B6D2F,. normal or B16F10 tumor-bearing. /iMCB for 48 h prior to washing, there was virtually no regrowth ' Days 1 and 7 or days 1, 4. and 7. even 6 days after reseeding in the absence of CB. /C57BL/6. * 1/1 (v/v) 95% ethanol/0.85% NaCl solution, pyrogen-free. TLC Analysis of Washed and Unwashed Cultures. To deter ' Drug administered over ventral wall of peritoneum except as noted. mine the effectiveness of the washing procedure, cell cultures ' Day 1 or days 1 and 4. were treated with CB and washed. CB levels in the original * Drug administered over the lateral wall of the peritoneum. treated medium, in the combined washings, and in the washed cells were determined by TLC analysis (see "Materials and Methods"). CB was detectable only in the original drug-treated or 3 days after drug treatment and was less than 5%. Drug suspension in CM-cellulose/Tween administered at 50 mg/kg medium, and the combined washings. It was below the limit of detection, that is less than 1 ^g of CB/106 cells, in the methanol i.p. to either normal or tumor-bearing mice was the maximum consistently tolerated i.p. dose (

100 parable in either of the treated groups, compared to controls (X) L (data not shown), but there was a marked increase in the duration of host survival in both of the treated groups (Fig. 4). Animals receiving 100 mg/kg CB on day 1 exhibited a median X60 survival of 28 days, compared to vehicle control survival of 17 days (165% survival treated/control). At 10 mg/kg CB, survival V 100 Mg/kg was 126% (treated/control) (P < 0.005). 40 D 10 M9/k9 In a separate experiment where animals received 150 mg/kg X Uel-iiole CB s.c. and were sacrificed 12 days after tumor implant to Z 20 permit direct measurement of tumor burden, there was also a substantial inhibition of tumor growth. The treated group had 10 15 20 25 30 35 tumors with mean weights of 159 mg, compared to vehicle Day controls which had mean tumor weights of 1.25 g. Fig. 3. Effect of CB s.c. on the time of B16F10 tumor nodule appearance. Cytochalasin B Effects on M109 Lung Carcinoma. Since nei Animals under nembuta! anesthesia were implanted with a solid piece of B16F10 tumor s.c. using a 13-gauge trocar. Treatment with CB in 2cc CM-cellulose/lÃ®r ther the B16F10 nor the B16BL6 lines exhibited spontaneous Tween 20 at 10 or 100 nig/kg or with vehicle alone was given s.c. peritumorally invasion and mÃ©tastasesfrom s.c. implants, we investigated the 1 day after tumor implant. Animals were considered positive for tumor nodule effects of CB in vivo on invasion and spontaneous metastasis appearance when a definite mass could be palpated. Either treatment was signif icantly different from vehicle controls (P < 0.005. n = 6). using the M109 lung carcinoma model. The pattern of growth and mÃ©tastasesof the Madison 109 lung carcinoma is depend ent upon the route of tumor injection. Table 2 presents the IODI? median survival times and the patterns of metastatic spread observed for the different routes of tumor challenge. Intraperi- toneal tumor spread more extensively and killed more rapidly than either i.v. or s.c. tumor challenge. The i.p. tumor spread

Table 2 Madison 109 lung carcinoma survival and mÃ©tastases Animals with one or more mÃ©tastasesLiver89 Tumor survival route"i.p. (days)22 of animals47

i.v.s.c.Median35 19 42 0 00Lung159552 32Number 58% 10Pancreas9616Spleen6 IO 15 OftV 25 30 48 Â°I x IO5viable M109 cells injected. Fig. 4. Effect of CB on host survival with B16FIO tumor s.c. Animals were challenged and treated as in Fig. 3. Animals were terminated when tumors exceeded 2.5 cm in any one dimension or when animals appeared to be in distress 4 1 or moribund. Median survival was significantly increased in the 100 mg/kg group (10) (percentage of median life span treated/control = 165; P < 0.005). >- CMC/TVEEN,DI

3 >- CB 150,01 doses, weight loss was minimal in animals surviving the acute initial response to CB. Multiple daily doses of CB were admin istered i.v. with no apparent cumulative toxicity (data not shown). Since there is a very sharp dose-response effect for the toxicity of CB administered i.v., the drug was tested for in vivo effects on tumor growth at 5 mg/kg, which is one half the maximum tolerated dose in CD2Fi mice. CB in CM-cellulose/Tween suspension at 150 mg/kg admin istered s.c. ventrally over the peritoneal wall was approximately u the LD,5 dose, and this dose could be used for in vivo testing, o but 100 mg/kg was more reliably tolerated. CB dissolved in DMSO and administered s.c. was also tolerated at 100 mg/kg, I- but only if injected laterally on the peritoneal wall. Injection in DMSO s.c. at 100 mg/kg ventrally over the peritoneum was relatively more toxic, presumably due to rapid penetration into the peritoneal cavity by the dissolved drug. Effects of CB on B16F10 Implanted s.c. CB administered as a single bolus dose at either 100 or 10 mg/kg s.c. under the tumor challenge site, 24 h after trocar implant of B16F10 solid tumor s.c., had marked effects on tumor latency, tumor growth 14 15 24 25 rate, and host survival. Fig. 3 shows the delay in the appearance of measurable tumor nodules in the treated groups compared DAY Fig. 5. Growth of s.c. M109 tumors in animals treated s.c. with vehicle (2% to vehicle (CM-cellulose/Tween 20)-treated controls. The me CM-cellulose/lco Tween-20) compared to animals treated s.c. with CB at 150 dian of tumor appearance in the controls was 7, whereas treat mg/kg single bolus dose 1 day after tumor challenge (1 x IO5viable cells) (A) or ment with CB at 10 mg/kg delayed the median appearance day at 100 mg/kg single bolus dose administered when tumors first became detectable (B). (TAD. tumor appearance day; 4 to 6 days after tumor implantation), n. to 13.5, and at 100 mg/kg to day 18 (P < 0.005). Once the number of animals per group. Growth rates of treated versus vehicle controls are tumor nodules began to grow, their growth rates were com significantly different, P< 0.001 in A: P< 0.01 in B (Student's t test). 1435

Table 3 Cylochalasin B s.c. versus Madison 109 lung carcinoma s.c. (1 x 10* viable cells) CBtreatment"mg/kgVehicle Lifespan weight at with survivors, (day)23 survival(%)30 T/C death(g)2.6 invasion9/10 day500/5 Â±0.5* 150Vehicle 1111 Z3282822-36'' 0.6 Â±0.2e4.9 1/10Â°4/5

Â±1.1 ISOVehicle 4.4 Â±2.26.4 1/33/5

1003T Â±0.3 ISOVehicle 11 36-38''28 12334 5.7 Â±0.34.9 2/34/5 0/30/42/6

Â±1.1 100Vehicle TAD7TAD 2829-36'' 3.8 Â±1.06.8 4/52/42/6Tumor-free

100 Â±1.0 100Day1 TADNecropsy 28-50''Median 40 118Tumor 3.7 Â±1.3Fraction " Vehicle. CM-cellulose, 1%/Tween 20, 2%. * Mean Â±SE. ' P = 0.001 compared to vehicle controls. ''Terminal. ' P < 0.05 compared to vehicle controls. 7TAD. tumor appearance day. primarily to peritoneal sites, whereas the i.v. injection produced VEHICLE,DAY 1 or TAD tumor colonies (artificial mÃ©tastases)in the lungs of all chal 100 lenged animals and in the livers of some of them. The s.c. CB: 100, TAD challenge metastasized mainly to the lungs. These results are -i ^ 80 consistent with the findings of other investigators (33, 39, 40). S.E.M. When the s.c. M109 tumor was treated s.c. with CB sus CD LU 7/9 pended in CM-cellulose/Tween, following procedures used for Z

Table 4 In vivocytotoxicity of cytochalasin B against BlfiFlO melanoma both to vehicle controls and to untreated controls. A significant Established tumors (8 days after trocar implant s.c.) were treated s.c. with 10 or 100 mg/kg CB or vehicle on day 8 post tumor challenge. Tumors were removed increase in the mean number of nodules in the liver was ob on either day 12 or day 14 (4 or 6 days after CB treatment), at which times there served in the animals treated with a single dose of CB on day was a significant inhibition of growth of tumor in the CB-treated group. Tumors 1, when compared to the controls, but in animals given CB from treated and control groups were analyzed individually for viability. Second ary recipients received 1 x 10*cells s.c. Three tumors were analyzed per group. treatment i.p. on days 1,4, and 7 the increase in liver mÃ©tastases None of the comparisons are significantly different at the 0.05 level. versus vehicle controls was not statistically significant. This viabil multiple-dose group showed a slight increase in survival (17%) ity(%)Â°TBE23 survival compared to vehicle controls. These effects of Â¡.p.CBat 50 mg/ GroupVehicle day12 TAD*7 (days)'17 kg are consistent with transient immunosuppression produced under these conditions7 and are not indicative of cytotoxicity 14 17 18 against the tumor challenge. CB, 10mg/kg 12 20 17 14 13 15 8 16 Effect of CB i.v. on M109 i.v. When CB was administered i.v. CB, 100mg/kgAnalysis 12 15 14 at 5 mg/kg in 48% ethanol either 1 hour prior to i.v. tumor 14Cell 15PE18 16Second 9Recipient 18 challenge (1 x IO5viable cells) or 24 h subsequent to i.v. tumor Â°TBE, Trypan blue exclusion; PE, plating efficiency. challenge, there was no increase in lifespan compared to vehicle- * TAD, median tumor appearance day of secondary recipient. c Survival, median survival (days). treated controls (7 or 8 animals/group; data not shown). All animals had multiple lung nodules when examined either at day 20 or at the terminal stage of the disease, and there was no conceivable following s.c. treatment under the tumor site, so it significant difference in the number of nodules in the treated was necessary to determine more directly whether CB could compared to the control groups. No mÃ©tastaseswere visible in function as a localized cytotoxic agent in vivo. Animals were the spleen, liver, or pancreas in any of the mice, indicating that challenged with B16F10 tumor s.c. and the tumors were allowed CB administered i.v. does not enhance splenic mÃ©tastases. to grow to measurable size (8 days), at which time they were treated with CB at either 100 or 10 mg/kg s.c. under the tumor site or with CM-cellulose/Tween 20 vehicle. Four or 6 days DISCUSSION subsequent to drug treatment, at which time growth inhibition More than 3000 papers dealing with the effects of CB in vitro was apparent, tumors were excised and the viability of the cells have been published, and some work has appeared on the in was determined by plating efficiency, dye exclusion, and in vivo vivo toxicity of other cytochalasin congeners (27-29). However, bioassay in recipient mice. Table 4 presents the results of this the work presented here is the first detailed in vivo evaluation study. It is apparent that cell viability is not compromised by of the toxicity of CB administered i.p., s.c., or i.v. and is the CB treatment, in comparison with vehicle control-treated cells. first in vivo evaluation of CB against a tumor model system. It In vivo bioassay of the cells recovered from treated tumors is apparent that against a s.c. tumor challenge a single dose of shows that the latency period prior to tumor nodule appearance CB administered s.c. at 100 mg/kg, which is below its LDi0, is in the recipients is not significantly affected in the CB-treated able to delay the time of tumor appearance, inhibit tumor donor tumors, nor is the survival of the recipient mice signifi growth rate, and prolong host survival in B16F10 melanoma cant different when they received tumor from groups treated and M109 lung carcinoma. The growth inhibition and extended either with high or low dose CB, as compared to those chal survival is true whether drug treatment is given 1 day after lenged with vehicle-treated control tumors (P > 0.05). tumor challenge or treatment is withheld until palpable tumor The absence of cytotoxic effect of CB in vivo is further nodules have appeared. In the M109 model system a single supported by experiments utilizing multiple bolus doses of CB dose of CB also inhibits tumor invasion and spontaneous lung administered i.p. at 50 mg/kg to animals bearing s.c. B16F10 mÃ©tastases.The effects on the B16F10 model persist at a 10- tumors.6 Systemic CB administered on days 1, 4, and 7 after fold lower dose, and at either dose it does not appear that effects s.c. tumor challenge produces a small but significant inhibition are due to measurable gross cytotoxicity. As shown in Table 4, of tumor growth rate compared to controls, suggesting that clonogenicity, tumorigenicity, and vital dye exclusion are not high, localized, potentially cytotoxic concentrations of CB are significantly altered by CB treatment in vivo, at times (4 and 6 not necessary for inhibition of growth of s.c. tumor nodules. days after CB treatment) when tumor growth is significantly Effects of CB i.p. on B16F10 or M109 i.p. The absence of inhibited in the treated animals. Evidence on CB clearance in direct cytotoxicity by CB on B16F10 melanoma cells in vivo is vivo (2)J indicates that CB would be cleared from the injection also apparent when CB at the maximum tolerated i.p. dose of site and from the animals long before the 4- to 6-day post- SO mg/kg is administered to animals bearing an i.p. tumor injection point, especially at the 10 mg/kg low dose treatment. challenge (Table 5). With CB treatment given either as a single Work with M109 lung carcinoma intradermal tumors treated bolus dose 1 day after tumor challenge or as two bolus doses with CB administered s.c. or i.v.* confirms this delayed effect on days 1 and 7, increase in survival compared to vehicle of CB on tumor growth, suggesting a noncytotoxic basis for the controls was less than 20%. CB under these conditions also action of CB. produced a marked enhancement of metastasis to the spleen at The mechanistic basis for the inhibition of growth and me the terminal stage of the disease and appeared to accelerate the tastasis remains to be established, but the effects of CB on seeding of mÃ©tastasesto the liver and pancreas as well as to the cytoskeletal structure and function suggest that alterations of spleen when necropsy was performed on day 12 after tumor either tumor or host cell movement could lead to measurable challenge. effects on local tumor growth, invasion, and metastasis. CB and As shown in Table 5, CB administered i.p. against a M109 other cytochalasin congeners have been shown to be noncyto i.p. challenge produced a marked increase in the proportion of toxic against a variety of cell types in culture at concentrations animals exhibiting splenic mÃ©tastasesand a significant increase up to 100 Mg/ml (200 MM)for short term exposures in the range in the mean number of nodules in the spleens, when compared 7 D. Bogyo and T. P. Fondy. unpublished observations. * McQuiggan and Fondy, unpublished observations. 8J. M. Mitchell and T. P. Fondy, unpublished observations. 1437

Table 5 Effect of cytochalasin B i.p. on BI6FIO melanoma and on M109 lung carcinoma i.p. dosemg/kgMI09, CB (nodules)Liver5/6 with terminal mÃ©tastases survival (days)20202224161614Fraction i.p.UntreatedVehicle505(1BI6F10,1 x 10*viable cells (10 Â±3)Â°5/6 (835/6 (0.2Â±2)1/6(1 1,411.4,71.71,71Median (10 Â±2)5/6(53 (885/6 Â±0.6)5/6 Â±17)6/6(21 17)Â±11)5/5(836/6 (12 Â±5)6/6 Â±5)3/5 (83Â±17)Â±13)Â± (12 Â±2)0/54/4(6.5 (2Â±1/5(0.41/4(0.32/4(1.81)Â±0.4)Â±0.3)Â±D i.p.Vehicle5050Day1;1 x 10' viable cells (2Â±1)4/4 (3 Â±0.5)4/4(154/4(16Â±2.2)Â± 0)4/4(100 Â± 1.8)4/4 Â± (87 Â±7.5)Pancreas5/6 3.8)Spleen1/6 (4.5 Â±0.9)Lung0/60/60/62/6 ' Mean Â±SE. * Vehicle. 2c.i carboxymethyl cellulose/1% Tween 20 in 0.85% NaCl solution. of 2 h (41). The profound pleotropic effects produced by CB on semisynthetic modifications of natural congeners is readily cells in culture depend on continued presence of the drug and accessible. are in many cases reversible upon removal of the CB (for detailed references see Ref. 2). The work presented here shows REFERENCES that CB can produce irreversible effects on cell proliferative ability with prolonged exposure in vitro. However, in addition 1. Vahara. I.. Harada, F.. Sekita, S., Yoshihira, K., and Natori. S. Correlation between effects of 24 different cytochalasins on cellular structures and cellular to the viability studies on B16F10 cells recovered from treated events and those on actin in vitro. }. Cell Biol., 92: 69-78, 1982. mice (Table 4), other lines of evidence argue against generalized 2. Lipski, K. M., McQuiggan, J. D., Loucy, K. J.. and Fondy, T. P. Cytochalasin B: preparation, analysis in tissue extracts, and pharmacokinetics after intra- cytotoxicity by CB in vivo. 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Peter F. Bousquet, Lesa A. Paulsen, Christopher Fondy, et al.

Cancer Res 1990;50:1431-1439.

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