[CANCER RESEARCH 52, 1721-1728. April I, 1992]

Benzoquinonoid Ansamycins Possess Selective Tumoricidal Activity Unrelated tosrc Kinase InhibitionLuke Whitesell,1Stuart D. Shifrin, Gisela Schwab, and L. M. Neckers

Tumor Cell Biology Section, Clinical Pharmacology Branch, National Cancer Institute, N1H, Bethesda, Maryland 20892

ABSTRACT

The benzoquinonoid ansamycin antibiotics herbimycin A and geldan-amycin have been shown to reverse the oncogenic phenotype of pp60v-src transformed cells as well as induce differentiation in a number of invitro model systems, reportedly due to their inhibition of src familyprotein tyrosine kinases. We now report that these agents are potentcytotoxins in vitro against a panel of highly malignant human tumor celllines possessing primitive neural features. Proliferation and/or survivalof fibroblasts, primary neuronal cultures, and several leukemia cell linesare unaffected at concentrations resulting in >99% cell loss in sensitivelines. The tumorigenicity in nude mice of sensitive cell lines can also bemarkedly reduced by either systemic or topical administration of theseagents without apparent toxicity to the whole animal. The cytocidalaction of these ansamycins is initiated very rapidly, is irreversible, and isclearly distinct from the delayed inhibition of src family kinases that hasbeen reported previously. Due to their potency, relative selectivity, andnovel mechanism(s) of action, these drugs could prove clinically useful inthe therapy of a number of human cancers of neural derivation.

INTRODUCTION

Benzoquinonoid ansamycin antibiotics were first isolated inthe late 1970s from the culture broths of several actinomycetespecies (1-3). Considerable interest was generated by theirunusual ansa bridge structure, and a number of compoundsincluding HA2 and GA were screened as possible antiretroviral

and antitumor agents. Results against the usual test cell linesof the time such as LI 210 leukemia and P-388 were disappointing. The concentrations required for antitumor activity werequite toxic in the whole animal, and further development wasabandoned. A new wave of enthusiasm was generated in thelate 1980s when it was noted that HA was able to revert thephenotype of tyrosine kinase oncogene-transformed cell linesat quite modest concentrations (4,5). Inhibition of angiogenesis(6) and induction of differentiation (7-9) in a number of modelsystems were also reported. Due to its apparent activity as amodulator of src family tyrosine kinases (10, 11), interest inHA has persisted, and reports continue to appear describingthe effects of the compound on various experimental cellularsignal transduction systems (12).

Targeting the developmental biology of particular tumorlineages appears to be a promising approach to the design oftruly tumor-specific chemotherapeutic agents. We have usedcell lines of the pediatrie cancers neuroblastoma, neuroepithe-lioma, and medulloblastoma as model systems in which toexplore the therapeutic potential of reagents able to modulatekey aspects of primitive neural development (13). Metastatic,locally disseminated, or recurrent pediatrie and adult cancers

Received 10/8/91; accepted 1/23/92.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at Pediatrie Branch,

National Cancer Institute, Building 10, Room 13N240, Bethesda, MD 20892.•¿�'The abbreviations used are: HA, herbimycin A; GA, geldanamycin; 2-ME, 2-

mercaptoethanol; MTT, 3-[4,S-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide; ICV. 1C,,,, concentration of drug resulting in a 95% or 50% decrease,respectively, in mean cpm of triplicate wells relative to untreated control wells.

of primitive neural derivation are among those malignanciesmost refractory to cure by current multimodality treatmentregimens. New approaches to treatment are clearly needed. Thebenzoquinonoid ansamycins may provide such a novel approachin that they appear to exploit unique properties of these refractory tumors. We now describe the selective spectrum of tumor-icidal activity in vitro for these compounds, evidence of in vivoan titumor activity, and data on the mechanism of their cytotox-icity which make it clear that the effects we describe are distinctfrom simple inhibition of tumor src family tyrosine kinaseactivity.

MATERIALS AND METHODS

Cell Cultures. CHP-100 cells were obtained from Dr. A. Evans(Children's Hospital of Philadelphia). The cell lines TC-32 and

NIH3T3 were obtained from Dr. M. Tsokos (National Cancer Institute,Laboratory of Pathology). All other cell lines used were purchased fromthe American Type Culture Collection (Rockville, MD). Primary neonatal rat cortical neurons were established using standard techniquesand supplied by Dr. M. Koenig (Armed Forces Research Institute,Washington, D.C.). All cell lines were tested using a Mycotect kit(Gibco Laboratories, Grand Island, NY) and were found free of Myco-plasma contamination. All culture media were supplemented with 10%fetal bovine serum (Whittaker Bioproducts, Walkersville, MD). Thecell lines CHP-100, TC-32, IMR-32, SKNSH, CEM, and HL-60 weregrown in RPMI 1640 (Biofluids, Inc., Rockville, MD). The cell linesD283 Med, D341 Med, SKNMC, SK-MEL-1, SK-MEL-2, and RPMI7951 were cultured in Eagle's minimal essential medium with 1%

nonessential amino acids and 1 HIMsodium pyruvate (all from Biofluids,Ine). NIH3T3 cells were cultured in Dulbecco's modified Eagle's me

dium with 4.5 g/liter glucose (Biofluids, Ine). Herbimycin A (NSC305978) and geldanamycin (NSC 122750) were obtained from the DrugSynthesis and Chemistry Branch, National Cancer Institute, formulatedas 2 mg/ml stock solutions in dimethyl sulfoxide (Sigma Chemical Co.,St. Louis, MO), and stored at 4°Cin the dark. For the lek studies,

peripheral blood lymphocytes were isolated from normal volunteers bystandard density centrifugation techniques. Cells were allowed to adhereto plastic overnight in RPMI 1640 containing 10% fetal bovine serum.Only nonadherent cells were used in the experiment.

Cell Proliferation and Survival Studies. Thymidine incorporationstudies were performed as previously described (14), except that serialdilutions of HA or GA were added to triplicate wells in 100 ^1 of theappropriate growth medium for the cell line being tested. Plates werecultured for 48-72 h and then pulsed with [me/A>'/-3H]thymidine (Du

pont, Boston, MA) for 4 h. followed by automated cell harvest andliquid scintillation counting. As an assay of relative viable cell number,mitochondria! reduction of MTT was used (15). Ninety-six-well tissueculture plates were set up as described above but cultured for 5-6 days.Twenty ¿ilof a 5 mg/ml solution of MTT (Sigma Chemical Co.) inphosphate-buffered saline was then added to all wells, and plates wereincubated for a further 4 h in the dark. The plates were centrifuged, themedium was removed, and 150 n\ of dimethyl sulfoxide (Sigma Chemical Co.) were added to each well. After a 10-min incubation in the darkwith shaking, the absorbence at 540 nm was determined (as a measureof the formazan concentration generated in each well) using a Bioki-netics plate reader (model EL-312; Bio-Tek Instruments, Inc., Wi-nooski, VT).

DNA Analysis. CHP-100 cells (5 x 106/10-cm tissue culture dish)

were plated in the presence of HA (500 nM), 80 nM cycloheximide (a1721

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concentration previously determined to inhibit protein synthesis by75% as measured by pHjleucine incorporation), or a combination ofthe two. Sixty-eight h post-plating the cells were harvested by trypsin-ization, and cell number and viability were determined by trypan blueexclusion in a hemocytometer chamber. The cells were washed once inphosphate-buffered saline, and high-molecular-weight DNA was prepared according to a previously reported method (16). Briefly, cellpellets were incubated at 37°Cfor at least 4 h in 0.5 ml of lysis buffer

(200 mivi Tris, pH 8.5, 100 mivi EDTA, 50 Mg/ml proteinase K, 1%sodium dodecyl sulfate). The DNA was then phenol extracted, and theaqueous phase was dialyzed overnight against 10 mM Tris, pH 7.5, lmM EDTA. The DNA was then incubated at 37"C with 50 ng/m\

RNase A. After 5 h, 120 Mg/ml proteinase K was added, and theincubation was continued for an additional 5 h. DNA was extractedwith phenol followed by chloroform and then precipitated with ethanol/sodium acetate. After redissolving in water, 5 tig of DNA were loadedper lane on a 1.4% agarose gel and electrophoresed for 16 h at 35 Vprior to staining with ethidium bromide and photography.

Immunoblotting. src protein expression was evaluated by immuno-blotting essentially as previously reported (14), except that after elec-trophoresis and transfer to nitrocellulose, detection of primary antibodywas achieved using the chemiluminescent alkaline phosphatase substrate AMPPD and alkaline phosphatase-conjugated goat anti-mouseimmunoglobulin (Western Light Kit; Tropix, Inc., Bedford, MA). Theire-specific mouse monoclonal antibody Mab 327 was obtained fromOncogene Science (Manhasset, NY) and has been described previously(17).

Determination of src Family-specific Kinase Activity. Cellular proteinextracts were analyzed for ire-specific kinase activity using a standardimmunoprecipitation protocol as described by Sartor et al. (18). Im-munoprecipitation was performed with Mab 327 (see above) as primaryantibody. The irrelevant mouse monoclonal antibody P3X63AG8 wasused as a control for the specificity of immunoprecipitation. /dt-specifickinase activity was determined using an anti/cA rabbit polyclonalantibody (a kind gift of Dr. A. O. Sartor, National Cancer Institute).In these experiments, rabbit preimmune serum was used as the antibodycontrol, src and lek autophosphorylation as well as ire- and /cAr-specificphosphorylation of exogenously added enolase substrate (Sigma Chemical Co.) were assessed using autoradiography. In some experimentscells were pretreated for varying intervals with HA or GA prior to lysisand immunoprecipitation. In other experiments, untreated cells werelysed and immunoprecipitated. HA or GA was then added to theimmunoprecipitates just prior to the initiation of the kinase reaction.

Tumorigenicity Studies. CHP-100 cells were harvested from subcon-fluent monolayers and resuspended at 5 x IO7 cells/ml in phosphate-

buffered saline. One hundred ^1 were inoculated s.c. into the right andleft inguinal areas of 6-week-old male athymic nude mice (FrederickCancer Research Facility, Frederick, MD) on day 0. Therapy with HAor GA was initiated either on the day of tumor inoculation or 10 dayspostinoculation when palpable tumors were apparent. The drugs wereadministered either topically by painting 5 M'of a 2 ing/ml solution ofthe drug in dimethyl sulfoxide onto the site of tumor cell inoculationor systemically by i.p. injection of drug formulated in dimethyl sulfoxide. Control animals received identical treatments with the appropriatevehicle alone. Animals were sacrificed 21 days after cell inoculation,and the well-encapsulated tumors were resected and weighed. All studies involving the use of mice were carried out under protocols reviewedand approved by the Animal Care and Use Subcommittee of theNational Cancer Institute.

RESULTS

Selective Cytotoxic Activity of HA and GA against PrimitiveNeural Cell Lines. Fig. \A demonstrates the potent growth-inhibitory activity of HA against the primitive neuroectodermalcell line CHP-100 as measured by inhibition of [3H]thymidine

incorporation after 2 days of culture with the drug. Note thatthis cell line is approximately 10-fold more sensitive in vitro tothe inhibitory activity of HA than the nontumorigenic mouse

fibroblast cell line NIH3T3. Fig. IB demonstrates a similarpattern of cell type selectivity for the related ansamycin GA,but GA is approximately 10-fold more potent than HA.

Experiments were also carried out with these two cell linesassessing the effects of HA and GA on survival rather thaninhibition of DNA synthesis. Automated analysis of MTTreduction by treated cells in a 96-well microtiter format allowedus to generate dose-response curves similar to those in Fig. 1(A and B), but the end point was now the relative number ofviable cells remaining after 5 days in culture. As Fig. 1 (C andD) demonstrates, concentrations of HA or GA that result incomplete cell loss with CHP-100 do not effect the fibroblastcell line. Relative potency and selectivity were found to besimilar by MTT analysis and thymidine incorporation analysis.The effect seen in MTT experiments was not simple inhibitionof growth. Values actually declined from the time of initialplating, indicating cell loss (data not shown). Microscopic examination of treated wells prior to analysis also confirmed thatonly cellular debris remained.

Having demonstrated that HA and GA are actually cytotoxicto CHP-100 cells, we next examined some of the characteristicsof this cytotoxicity. Cell death can be an active or passiveprocess, depending on cell type and initiating stimulus. DNAdegradation is often used as an end point for both processes. Arequirement for ongoing protein synthesis in the transductionof cell death suggests an active type of program. Fig. 2 demonstrates that protein synthesis is necessary for HA-inducedcell death. In this experiment, cells were cultured in the presenceof 500 nM HA with or without the nonspecific protein synthesisinhibitor cycloheximide. After 68 h, cells were harvested andassayed for viability, and high-molecular-weight DNA was prepared. The viability of untreated cells was 82%, that of 80 HMcycloheximide-treated cells was 57%, that of HA-treated cellswas 26%, and that of HA/cycloheximide-treated cells was 66%.Cycloheximide treatment clearly inhibited the cytotoxic activityof HA. Analysis of high-molecular-weight DNA revealed substantial DNA degradation in the HA-treated cells (Fig. 2, LaneHA), but the nucleosomal cleavage characteristic of apoptosiswas not seen. However, DNA degradation was abrogated bycoincubation of the cells with cycloheximide (Lane CHX/HA).The quality of high-molecular-weight DNA in that lane isindistinguishable from that of either untreated (Lane CTRL) orcycloheximide alone-treated cells (Lane CHX). These findingsconfirm that HA is cytotoxic to sensitive cells and that thiscytotoxicity requires active cellular participation.

To further evaluate the spectrum of activity displayed by HAand GA, we screened a panel of primitive neural cell lines usingboth inhibition of ['H]thymidine incorporation and reduction

of MTT as end points. Table 1 is a summary of dose-responsedata obtained by thymidine assay for a variety of both neuraland nonneural cell lines. Nonneural cell types such as thehematopoetic lines HL-60 and CEM were quite resistant togrowth inhibition by HA and GA. All primitive neuroectodermal cell lines examined to date, both peripheral nervous system-derived (e.g., CHP-100, SKNMC, TC-32) and central nervoussystem-derived (e.g., D283 Med, D341 Med) have proved to bequite sensitive to HA and GA. Interestingly, cells from aprimary culture of the malignant pleural effusion of a patientwith relapsed, heavily pretreated Ewing's sarcoma were also

sensitive to HA, as determined by MTT assay (IC95= 400 HM).Cells of more mature neural phenotype were relatively insen

sitive to the cytotoxic activity of HA. Thymidine incorporationdata for the neuroblastoma cell lines IMR-32 and SKNSH are

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120

Fig. 1. Dose-dependent inhibition of cellproliferation and survival by benzoquinonoidansamycins. O, cell line ( III' 100; •¿�cell line

NIH 3T3. A, HA added to culture medium.[3H]Thymidine incorporation was assayed 48h later. B, GA added to culture medium. I'll]

Thymidine incorporation was assayed 48 hlater. C, HA added to culture medium. MTTreduction was assayed 5 days later. D, GAadded to culture medium. MTT reduction wasassayed S days later. Data are expressed aspercentage cpm or absorbance relative to control wells plated simultaneously without drug.All points represent the mean of triplicate determinations. SDs are less than 10%.

10 100Herbimycin A Concentration (ng/mL)

1 10 100 1000Herbimycin A Concentration (ng/mL)

120

1 10Geldanamycin Concentration (ng/mL)

i 10GeldanamycinConcentration(ng/mL)

100

shown in Table 1. The highly differentiated rat pheochromo-cytoma cell line PC-12 was also relatively insensitive to HA byMTT assay (IG,5 > 500 nivi). Fig. 3 demonstrates that primarycultures of neonatal rat cerebellar neurons were not affected byculture in 472 nM HA or 40 nM GA. This experiment was alsorepeated several times with neonatal rat cerebral cortical neurons with the same results. Lastly, the melanoma cell lines SK-MEL-1, SK-MEL-2, and RPMI 7951 were also screenedagainst HA by MTT assay. The one line found to be sensitive,RPMI 7951 (HA IC50 = 125 HM), is reported to possess themost primitive, least differentiated phenotype as defined byHoughton et al. (19).

Cytotoxicity Distinct from Direct Inhibition of src KinaseActivity. In order to evaluate the mechanism by which HA andGA induce cell death in sensitive cell lines, we first exploredthe time course of HA action. For precise definition of theduration of exposure to drug required for subsequent growthinhibition, we took advantage of the previously reported abilityof sulfhydryl compounds to rapidly inactivate HA (10). Thisinactivation strategy was felt to be superior to washout experiments in that drug could be rapidly and completely neutralizedwithout manipulation of the treated cells. In preliminary experiments we confirmed that sulfhydryl compounds such as 2-MEand dithiothreitol effectively inactivate HA. The growth ofCHP-100, while almost completely inhibited by culture in 118nM HA, was not affected by 500 HMHA that had been prein-

cubated with 100 ^M 2-ME for 30 min. Additional time course

experiments established that, indeed, the cytotoxicity of HAsteadily declines upon exposure to 2-ME until, after 10 min of

exposure, the drug is completely inactivated (data not shown).With this information in hand, we proceeded to determine theminimal time of exposure to active HA necessary to causegrowth inhibition. Fig. 4 depicts the results of a representativeexperiment in which CHP-100 cells were incubated in HA forvarying amounts of time prior to addition of 2-ME. Knowingthat 2-ME completely inactivates HA within 10 min, we determined, from 10 min onward, the effect of varying a cell's

exposure to active HA on its subsequent proliferative capacityas measured by ['H]thymidine incorporation 24 h later. Expo

sure to active HA for the shortest time interval (10 min) resultsin an 80% inhibition of DNA synthesis. Complete inhibition isobserved following a 1-h exposure. Clearly, whatever the mech

anism responsible for HA cytotoxicity, the process is rapid andirreversible.

The benzoquinonoid ansamycins have been reported to directly inhibit src family tyrosine kinases either by inducingdegradation of the enzyme or directly inhibiting its activity (11).Knowing the time course over which HA exposure induces celldeath, we examined possible effects of HA on src expression insensitive cells. Fig. 5 demonstrates by immunoblot analysis thatthe level of src protein in treated cells shows no modulation

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Fig. 2. Cycloheximide inhibits HA-induced DNA degradation. CHP-100 cellswere treated as indicated above each lane for 68 h. CTRL, no drugs added; //.-),500 nM HA added; CHX, 80 nM cycloheximide added; CHX/HA, both drugsadded simultaneously. DNA was isolated and electrophoresed as described in"Materials and Methods." Degradation of high-molecular-weight DNA is appar

ent only in Lane HA.

after up to 24 h in culture with 472 nM HA, a time at whichgrowth inhibition is complete.

Given that protein expression remains constant, we nextexamined c-src-specific kinase activity in HA-treated CHP-100cells. Fig. 6A depicts the results of an in vitro kinase assay withimmunoprecipitates prepared from cells treated for varyingintervals with 500 nM HA. In the even-numbered lanes, celllysates were immunoprecipitated with an irrelevant, isotype-matched control antibody. These lanes demonstrate the srcspecificity of the assay conditions used. Comparison of Lanes1 and 5 to Lanes 3 and 7 makes clear that there is no decreasein src autophosphorylation (S) or ire-mediated phosphorylationof the exogenously added substrate enolase (E) induced byculture of cells for 30 or 60 min, respectively, in HA, eventhough these durations of exposure to drug are sufficient toirreversibly inhibit subsequent DNA synthesis (see Fig. 4). Fig.6B depicts a similar experiment, but here cells were culturedfor 2 h prior to lysis and immunoprecipitation. Lane 1 containsimmunoprecipitate from cells treated with 100 nM GA (approximately 5 times the maximally effective cytotoxic dose). Lane 2contains immunoprecipitate from untreated cells (control), andLane 3 contains immunoprecipitate from cells treated with 1fiM HA (approximately 5 times the maximally effective cytotoxic dose). Again, no effect of treatment on src autophosphorylation or enolase phosphorylation is noted. Finally, Fig. 6Cdemonstrates that even after S h of incubation in HA, no effectis seen on c-src in vitro kinase activity. We have observed similar

results in two other sensitive neuronal cell lines, SKNMC andTC32 (data not shown), and in the insensitive cell line 3T3 (seeFig. 9>A,Lanes 1 and 2). As might be expected from theseresults, we also find that total cellular phosphotyrosine contentas well as the pattern of tyrosine-phosphorylated proteins inHA-treated cells is not significantly different from that ofuntreated cells at multiple time points (up to 4 h) when examined by antiphosphotyrosine immunoblotting (data not shown).

In a final attempt to demonstrate some effect of HA on thesrc kinase activity isolated from sensitive cells, we added 400nM HA (>1500 times the maximally effective concenti at ion onintact cells) directly to the in vitro immune complex kinasereaction mixture. Fig. 7 demonstrates that minimal inhibitionof enolase phosphorylation and no inhibition of src autophosphorylation were observed. To ensure that our negative findingsdo not represent a technical artifact of the kinase assay procedure used, we examined a system where HA has recently beenreported to down-regulate the activity of the src family kinaselek. As June et al. described (12), we found that culture in 1 ^MHA for as little as 4 h markedly reduced the /c&-specific kinaseactivity immunoprecipitated from peripheral blood lymphocytes (Fig. 8). Interestingly, exposure to 1 pM HA for up to 78h was not cytotoxic, and following drug washout, cells stillproliferated in response to the T-cell mitogen phytohemagglu-tinin. Additionally, as described previously (4, 5, 10, 11), wewere able to demonstrate that treatment of v-src-infected 3T3cells for 5 h with 0.5 ¿tMHA resulted in a significant decreasein immunoprecipitable src kinase activity as measured by immune complex assay, although identical treatment of uninfected3T3 cells had no such effect on c-src kinase activity (Fig. 8).Thus, while the mechanism(s) by which HA and GA inhibit cellproliferation and induce cell death in primitive neuroectoder-mal cell lines remains unknown, it appears to be clearly distinctfrom the direct effects on .vrffamily kinase activity described inother systems.

In Vivo Antitumor Activity. The utility of benzoquinonoidansamycins as potential chemotherapeutic agents in the treatment of selected human cancers was evaluated using a tumorxenograft/nude mouse model. These experiments must beviewed as very preliminary in that no pharmacological information regarding absorption, distribution, or metabolism ofHA and GA was available. Thus, the actual concentration ofactive drug to which tumor xenografts were exposed with thevarious dosing techniques is unknown. Although formal toxi-

Table 1 Selectivity of growth inhibition by HA and GAData are derived from dose-response curves generated by quantitation of |3H]

thymidine incorporation by indicated cell lines 48 h after plating in variousconcentrations of the indicated drug. Cell lines are grouped in decreasing orderof sensitivity.

DrugHerbimycin

A(nM)Cell

lineCHP-100TC-32SKNMCD283

MedD341MedIMR-32SKNSHNIH3T3HL-60CEM1C»6231626210236236710>472>4721C«23623623623630>472>4723780>472>472Geldanamycin(nM)1CM558ND°NDNDND80>40>401C,,152025NDNDNDND250>40>40

' ND, evaluation not done.

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Control Geldanamycin Herbimycin A

Treatment

Fig. 3. Lack of benzoquinonoid ansamycin toxicity to primary neurons inculture. HA at 472 MMor GA at 40 n\i was added to established neonatal ratcerebellar neuron cultures. Plates were incubated for a further 4 days, andreduction of MTT dye reflecting viable cell number per well was then assayed asabsorbance at 540 nm. Height of columns, mean absorbance of quadruplicatewells; bars, SD of the mean.

40000

30000 -

co o.

10000 -

0-

NoHerbl mycln A Added

10 100

Duration of Exposure to Active Herbimycin (min)

Fig. 4. Growth-inhibitory action of HA is rapid and irreversible. CHP-100cells were cultured in 500 nvi HA for the time periods indicated prior toinactivation of the drug by addition of 2-ME. [3H]Thymidine incorporation wasassessed 24 h later. Points, mean of triplicate determinations. The SD of eachpoint is less than 10%. (No data points are shown between 0 and IO min because2-ME requires this interval to completely inactivate HA under the conditionspresented).

cological evaluation and determination of lethal dose have yetto be performed, preliminary experiments in athymic rats havedemonstrated that i.v. infusion of 4 mg/kg (24 mg/nr) of GAis well tolerated, with no overt immediate or delayed toxicity.Table 2 depicts the effects of either topical or systemic administration of drugs to animals at the time of tumor cell inoculation. Topical therapy consisted of applying 5 ^1 of a 2 mg/mlsolution of drug in dimethyl sulfoxide to the skin overlying thetumor inoculation site daily for 5 days. Intraperitoneal therapyconsisted of GA injection daily for 5 days at 4.5 mg/kg bodyweight. A marked reduction in subsequent tumor formationwas evident following both HA and GA administration. The

mean weight of tumors that did form in drug-treated animals

was also significantly smaller. No overt toxicity as evidencedby weight loss, decreased motor activity, or local skin reactionwas noted. On one occasion, the tumors which did form despitetopical GA treatment were resected aseptically, disaggregatedin tissue culture medium, and grown for 9 days in vitro in thepresence or absence of GA. The cell cultures recovered in thisfashion grew well, and, importantly, despite previous in vivotreatment with GA they remained fully sensitive to its cytotoxicaction in vitro. This finding suggests that the failure of topicalGA to inhibit tumor formation in all animals was a result ofinadequate exposure to active drug and not acquired tumor cellresistance.

The ability of HA to inhibit the growth of established tumorswas also examined in the nude mouse model. Initiation of HA

1 23456

""***^<« SRC

Fig. 5. Immunoblot analysis of src protein expression. CHP-100 cells wereplated in the presence or absence of 472 nw HA. Protein lysates were preparedfrom replicate plates at several intervals post plating. Lane 1, lysate preparedfrom untreated cells just prior to plating; Lanes 2 and 3, lysate prepared fromHA-treated and untreated cells, respectively, after l h in culture; Lanes 4 and Õ,treated and untreated, respectively, after 18 h; Lanes 6 and 7, treated anduntreated, respectively, after 24 h.

-s

«» *» - •¿�»-E

B

-

-S

~» -E

Fig. 6. CHP-100 ire kinase activity is not affected by HA and GA as determinedby immune complex assay. A, early time points, immunoprecipitation from CHP-100 cells cultured with 500 RM HA. Lane 1, 30 min, untreated cells; Lane 3, 30min, cells treated with HA; Lane 5, 60 min, untreated; Lane 7, 60 min, cellstreated with HA; Lanes 2, 4, and 6, immunoprecipitation with irrelevant controlantibody as specificity control at 0, 30. and 60 min post-plating, respectively. /(.2-h time point. Lane I, 100 IIMGA; Lane 2, no drug; Lane 3, l /IM HA. C, 5-htime point. Lane I, no drug; Lane 2, 250 DM HA 5, src; E, exogenously addedsubstrate enolase.

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1

SRC

EMÓLASE

Fig. 7. Minimal in vitro inhibition of src kinase activity by HA. UntreatedCHP-100 cells were lysed and immunoprecipitated with Mab 327. HA was addedto one-half of the immunoprecipitate to achieve a concentration of 400 »M(Lane/). The other half received an equal volume of dimethyl sulfoxide vehicle (Lane2). Extent of subsequent src autophosphorylation <.S>and phosphorylation ofexogenous substrate (/•")are indicated.

result in rapid and complete cell death have been reported bymany previous investigators to be nontoxic in a variety ofnontumorigenic cell lines and primary cultures (11,12, 20). Wealso found little toxicity on primary neuron cultures, severalhematopoetic cell lines, and a mouse fibroblast line. The reasonfor the restricted pattern of tumoricidal activity remains unclearat this time. We are currently examining the possibility thatcertain cells are insensitive to HA and GA because they rapidlyinactivate extracellular drug through redox (10) or DT-dia-phorase (21-23) pathways. We are also attempting to developresistant subclones of sensitive lines via standard dose escalation techniques. Resistant clones should provide a clue tounderstanding the restricted pattern of activity observed andhelp define the potential for development of clinically significant drug resistance.

An alternative hypothesis to explain the restricted pattern ofcytotoxicity we have observed is that sensitive tumor cells arefrozen in a particular stage of neural development (due to theironcogenic transformation), where an active program of celldeath can be triggered by the ansamycins. Much evidence existsfor programs of active cell death within developing systems (24,

B

vSRC- LCK-

1 2 1 2 12345Fig. 8. HA treatment decreases lek and v-src kinase activity but not c-src kinase

activity as determined by immune complex assay. .I. Mab 327 immunoprecipita-tion from lysates of NIH 3T3 cells. Lane I, cells treated for 5 h with 500 n\i HAprior to lysis; Lane 2. untreated cells. //. Mab 327 immunoprecipitation fromlysates of NIH 3T3 cells stably transfected with \-src construct. Lane 1, cellstreated for 5 h with 500 n\i HA prior to lysis; Lane 2, untreated cells. C anti-tatimmunoprecipitation from peripheral blood lymphocytes. Lane 1, control precipitation with preimmune serum; Lane 2, untreated cells; Lane 3, cells treated for4 h with 1 j<\i HA, washed, and incubated for an additional 14 h without drugprior to lysis; Lane 4, cells incubated for 14 h prior to addition of 1 n\i HA andlysis 4 h later: Lane 5, cells treated for 18 h with 1 pM HA prior to lysis. /.exogenously added substrate enolase.

treatment 10 days after tumor cell inoculation at a dose of 1.5mg/kg, given i.p. every third day for four doses, resulted in

Table 2 HA and GA decrease the tumorigenicity of CHP-100 in vivo

Nude mice were inoculated with tumor cells s.c., and therapy was begun onthe same day. Topical therapy consisted of application of 10 i/n of drug to theskin overlying the tumor inoculation site daily for 5 days. Systemic therapyconsisted of i.p. injection of 90 jig GA (4.5 mg/kg) daily for 5 days. Tumorformation as defined by resection of a discrete mass of 100 mg or greater wasassessed 21 days after cell inoculation.

Topical Systemic

Drug TumorsMean weight

(mg) TumorsMean weight

(mg)Dimethyl sulfoxide control 10/10" 357(49)* 10/10 436(93)Geldanamycin 5/10 302f (97) 6/10 2S2*(53)HerbimycinA 7/10 221'(49) ND^ ND

" Number of tumors formed/number of sites inoculated.* Numbers in parentheses, SE of the mean value.c Comparison to control by Student's I test performed on log-transformed

data, P = 0.037.•¿�*As above, P = 0.042.' As above, P = 0.014.•¿�^ND,experiment not performed.

2.0-

edecreased tumor mass as determined on the 21st day after Ktumor cell inoculation (Fig. 9). <2

DISCUSSION

Metastatic and locally disseminated cancers of primitiveneural derivation remain among the most refractory to curewith current multiagent treatment regimens. While exploringdrug-induced differentiation of model tumor cell lines as atherapeutic approach in these malignancies, we became awareof reports that the benzoquinonoid ansamycins were able toinduce or augment differentiation (7-9). In contrast to theeffects reported, however, we found that these drugs possessvery potent cytocidal activity in vitro against a select range ofhuman tumor lines. The concentrations of HA and GA that

1.0

0.0

VEHICLE HERBIMYCIN AFig. 9. Systemic treatment with HA of tumor-bearing mice inhibits tumor *

growth. Nude mice with established s.c. tumors received i.p. injections of either1.5 mg HA/kg or an equivalent volume of vehicle on days 10, 13, 16, and 19after tumor cell inoculation. Points, weight of an individual tumor mass resectedon day 21. , mean weight of tumors resected from the vehicle-treated mice.Comparison of control to HA-treated tumor weights by Student's l test on log-transformed data yields P = 0.057.

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25), and, in fact, selective cell loss probably represents a majormechanism for the establishment of specific cell-cell interactions within the nervous system. Our findings that HA-inducedcell death correlates with rapid degradation of high-molecular-weight DNA and that both of these phenomena can be blockedby inhibition of protein synthesis are supportive circumstantialevidence for induction of an active program of cell death (although probably not apoptosis in the strict sense) by these drugsin sensitive cells.

Is src involved in mediating HA/GA cytotoxicity? Althoughthe sensitive cell lines we have discussed express enzymaticallyactive src protein as determined by Western blot analysis andin vitro kinase assay (26), insensitive cells (i.e., NIH3T3, PC-

12) also express src protein and tyrosine kinase activity (datanot shown). Expression of neuronal isoforms of src (27) doesnot appear to correlate with sensitivity to these drugs. Thesensitive, primitive neuroectodermal cell line SKNMC expresses only nonneuronal src (28), while insensitive rat braincortical and cerebellar primary cultures express high levels ofneuronal src (29). Additionally, the relatively insensitive neuroblastoma SKNSH expresses high tyrosine kinase activity andat least two isoforms of src (28).

HA has been reported to inhibit src and src family kinasesand to stimulate the degradation of these enzymes when addedto cells in culture. Although one study found that as little as 1h of exposure of Rous sarcoma virus-transformed normal ratkidney cells to 1 n\i HA significantly reduced v-src kinaseactivity (11), others have found no change in either cellularphosphotyrosine content or src family kinase activity until 8-12 h after cell exposure to HA (12). However, since inhibitionof src or src family kinases by HA has been associated withgrowth inhibition and reversion of transformed cells to normalphenotype, we explored the possible involvement of src in theHA-induced cytotoxicity observed in CHP-100 cells. Our results do not implicate a direct decrease of src kinase activity inthe response of these cells to HA. No changes in total cellularphosphotyrosine, src protein level, or src kinase activity wereobserved in treated cells over a period of exposure extendingwell beyond that required for induction of subsequent cell death.Moreover, in concordance with a recent report (12), we did notfind HA able to directly inhibit src kinase in vitro, except atconcentrations much greater than those required for cytotoxicity on intact cells (Fig. 7). Nevertheless, because HA is ableto revert the morphology of Rous sarcoma virus-transformed3T3 cells (10), src-related pathways may still be involved in theselective cytotoxicity we have observed. In preliminary datafrom our laboratory, HA has been found to revert the morphology of v-src-transformed 3T3 cells at concentrations whichonly minimally affect levels of immunoprecipitated src auto-phosphorylation or phosphorylation of the exogenous substrateenolase. However, the pattern of associated proteins coimmu-noprecipitated with anti-src antibody in these cells is altered byprior treatment of the intact cells with HA (data not shown).Thus, HA and GA may act by interfering with interactions ofsrc and specific phenotypic or developmentally regulated targetsubstrates. Such protein-protein interactions mediated by so-called src homology domains (SH2, SH3) are critically important in modulating growth-regulatory signal transduction pathways (30). Interruption of SH2 or SH3 interactions by HA andGA would reconcile the previously reported activities of thesedrugs with the novel findings described here. Experimentsevaluating this hypothesis are currently in progress.

Because HA and GA appear to be selectively cytocidal, it is

of great interest to define their precise mechanism of actionfrom the points of view of both basic biology as well as clinicaldrug development. For example, understanding the mechanismof action of these drugs may allow for identification of othersensitive tumor types. We are continuing to evaluate melanomacell lines as an approach to defining the developmental/phe-notypic specificity of these ansamycins. In addition, we arescreening a panel of G A derivatives for potency and selectivityin our tissue culture model systems. The structure-activityrelationships generated should pinpoint the critical featuresrequired for activity and may suggest likely intracellular targetsfor these drugs.

Finally, the xenograft tumor results we report suggest promising, if restricted, in vivo antitumor activity for the benzoqui-nonoid ansamycins. The detection of any antitumor activity atall in vivo is quite encouraging given our nearly complete lackof baseline pharmacological information. The antibiotics discussed here are small, lipophilic molecules that should berelatively easy to produce in bulk and, following systemic administration, should readily penetrate solid tumors and eventhe blood-brain barrier. Compounds such as these may wellserve as models for the development of a generation of noveldrugs possessing both biological specificity and tumoricidalpharmacology.

ACKNOWLEDGMENTS

We thank A. Oliver Sartor and M. E. Horowitz for discussion andthoughtful review of this manuscript. We also thank K. L. Rinehart forhelpful discussions.

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1992;52:1721-1728. Cancer Res   Luke Whitesell, Stuart D. Shifrin, Gisela Schwab, et al.  

Kinase InhibitionsrcActivity Unrelated to Benzoquinonoid Ansamycins Possess Selective Tumoricidal

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