Growth by Disrupting Mitochondria

2 downloads 0 Views 1MB Size Report
Jul 1, 1995 - Angelika M. Burger, Gurmeet Kaur, Michael C. Alley, et al. ... obtained from the repository of the National Cancer Institute in vitro cancer screen.
Tyrphostin AG17, [(3,5-Di-tert -butyl-4-hydroxybenzylidene)-malononitrile], Inhibits Cell Growth by Disrupting Mitochondria Angelika M. Burger, Gurmeet Kaur, Michael C. Alley, et al. Cancer Res 1995;55:2794-2799. Published online July 1, 1995.

Updated Version

Citing Articles

E-mail alerts Reprints and Subscriptions Permissions

Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/55/13/2794

This article has been cited by 9 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/55/13/2794#related-urls

Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected].

Downloaded from cancerres.aacrjournals.org on July 12, 2011 Copyright © 1995 American Association for Cancer Research

[CANCERRESEARCH55, 2794—2799, July 1, 19951

Tyrphostin AG17, [(3,5-Di4ert-butyl-4-hydroxybenzylidene)-malononitrile], Inhibits Cell Growth by Disrupting Mitochondria Angelika M. Burger,' Gurmeet Kaur, Michael C. Alley, Jeffrey G. Supko, Louis Maispeis, Michael R. Grever, and Edward A. Sausville2 PR!/DynCorp. Frederick Cancer Research and Development Center, Frederick. Maryland 21 701 (A. M. B.), and Laboratories of Biological Chemistry (G. K., E. A. SI and Pharmaceutical Chemistry (J. G. S.. L MI, Biological Testing Branch (M. C. A.J and Office ofAssociate Director (M. R. G.J, Developmental Therapeutics Program. Division of Cancer Treatment, National Cancer Institute, NIH. Bethesda. Maryland 20892

ABSTRACT

investigators have further characterized AG17 as a potent inhibitor of EGF-stimulated pancreatic carcinoma cell growth (5), lectin, and [(3,5-Di-tert-butyl-4-hydroxybenzylidene)-malononltrile) (AG17), a IL-2-stimulated lymphocyte proliferation (1 1). “tyrphostin―tyrosine kinase antagonist, was found to inhibit tumor cell In considering potential mechanisms for the antiproliferative action growth with 50% growth inhibition ranging from 0.7 to 4.0 @M in a panel of AG17, it came to our attention that AG17 was identical in structure of 13 humantumorcell lines,as evaluatedby tetrazoliumdye reduction and inhibitionof precursorincorporationintomacromolecules.The pro to SF 6847, previously demonstrated in isolated rat heart and liver mitochondria to function as an uncoupler of oxidative phosphoryla myelocytic leukemia cell line HL-60(TB), was the most sensitive with tion (12, 13). We therefore undertook this study in an effort to clarify irreversible total growth inhibition after 12 h of exposure to 1.5 pM drug. Antiproliferative effects of AG17 in HL-60(TB) cells were temporally whether AG17 functioned primarily as a protein kinase antagonist or related to disruption of mitochondrial function, which occurred within I h mitochondrial poison when used in cell cultures at growth inhibitory after drug exposure as demonstrated by a significantly decreased mass of concentrations. We conclude that in the most sensitive human tumor ATP in drug-treated cells, loss ofthe fluorescent mitochondrial membrane cell line studied, HL-60(TB), AG17 acts as a potent inhibitor of potential probe rhodamine 123, and ultrastructural examination of mito mitochondrial function. The effect occurs within 1 h after drug addi chondria using fluorescence and electron microscopy. Specific decreases tion and appears to explain its antiproliferative activity in HL-60(TB) oftotal or tyrosine-phosphorylated substrate at concentrations ofthe drug notaffectingAlP levelswerenotdetected.Thesedataraisethepossibility and other cell lines studied in greater detail (jarticularly chronic myelogenous leukemia K-562). Although our results do raise the that AG17 may act in part by altering mitochondrial function and/or structure,andthatimpairmentof mitochondrialfunctionmaybe exploit possibility that AG17 may modulate mitochondrial function in a manner that could lead to a novel therapeutic approach, these findings able as a potentially useful mechanism to modulate tumor cell prolifera tion. This study also emphasizes the importance ofevaluating carefully the definitely emphasize the importance of investigating potential cellular effects of potential protein kinase antagonists, since these structures have mechanisms of growth inhibition for this series of compounds, in effects in intact cells in addition to what might be expected from in vitro addition to those effects which have been observed with purified enzyme assays. enzymes. INTRODUCTION MATERIALS Tyrphostins were originally described as a group of low molecular weight protein tyrosine kinase inhibitors designed to compete with substrate rather than AlT (1, 2). Tyrphostins include three structurally diverse classes of compounds: the hydroxy-cis-benzylidenemalononi true group, lavendustin derived-blockers, and bisubstrate quinoline analogues (3). These agents can clearly function as specific inhibitors of receptor tyrosine kinases (2, 4, 5) and non-receptor protein tyrosine kinases such as p2lO@@1 (6, 7), p185bCrabI, and p14@jabI(6). In a number of instances, inhibition of cell proliferation or function cor relates with inhibition of tyrosine kinase activity (6, 7). AG173 (Fig. 1), was found in a screening study to be the most potent tumor cell growth inhibitor among a series of tyrphostins examined as inhibitors of breast carcinoma cell growth (8). Prior studies with AG17 had demonstrated that it could function as an inhibitor ofthe EGF receptor tyrosine kinase (4). Evaluations in intact cells have revealed that AG17 is a potent inhibitor of PDGF-stimulated growth of rabbit vascular smooth muscle cells (9) as well as PDGF-mediated tyrosine phosphorylation

of several

intracellular

substrates

(9,

10).

Other

AND METHODS

Drug. AG17 (NSC 242557; Fig. 1) was provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD) and by Professor Levitzki,

Hebrew

University

(Jerusalem,

nide m-chlorophenylhydrazone,

Israel).

Antimycin

A, carbonyl

cya

and oligomycin were purchased from Sigma

Chemical Co. (St. Louis, MO). Stock solutions were prepared in DMSO [American

Burdick

and The Jackson

Laboratory

(Spectrophotometric

Grade

Product 081), Muskegan, MI]. Cell Culture and Cell Growth Assays. Culture medium and supplements were purchased from GIBCO-BRL Life Technologies, Inc. (Gaithersburg,

MD) and Costar Corp. (Cambridge, MA). All human tumor cell lines were obtained from the repository of the National Cancer Institute in vitro cancer screen. Cells (2 x 10@/well) were plated in 96-well plates in 100 @xl complete medium (RPMI 1640 supplemented with 2 mM L-glutamine and 10% fetal

bovine serum). After 24 h, 100 @d of drug solutions (prepared at twice the final concentration

using medium containing 0.5% DMSO) were added in increas

ing concentrations to a final volume of 200 s.d/well.Growth of drug-treated cells was compared to vehicle control-treated cells and quantitated after 6 days by MTI' reduction

as described

previously

(14, 15). The formazan

product of

Mfl' reduction was washed twice with PBS using the Costar Transplate Received 11/3/94; accepted 4/24/95.

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 18 U.S.C. Section 1734 solely to indicate this fact.

in accordance with

I Supported in part by the Deutsche Forschungsgemeinschaft. 2 To

whom

requests

for

reprints

should

be

addressed,

at:

National

Cancer

Institute,

NIH, EPN 843, Bethesda, MD 20892. 3 The

abbreviations

used

are:

AG17,

[(3,S-di-ten-butyl-4-hydroxybenzylidene)-mal

ononitrilel; EGF, epidermal growth factor; PDCIF, platelet-derived growth factor; GI@,

50% growth inhibition; TGI, total growth inhibition; P'l'K, protein tyrosine kinase; MTI', 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium

bromide; IL-2, interleukin

2;

Cartridge pipetter, dissolved in 150 @xlDMSO, and absorbance was measured with a Dynatech MR 5000 plate reader (Dynatech Laboratories Corp.,

Chantilly, VA) at 540 nm. The GI50 drug concentration and the TGI drug concentration were calculated as reported elsewhere (16). Reversibility of AG17 Effect. To determine the reversibility of AG17 in inhibiting cell growth, 20,000 cells/2 ml in a 35-mm Petri dish were exposed to drug (0.25—16ILM)24 h after plating. Cell recovery was assessed in assays

of 6-day duration following the removal of the drug at intervals from 1h to 6 days. Cells from each dish were subjected

to two medium

washing

steps in

tubes and subsequent cultivation for 6 additional days in drug-free medium.

HL-60(TB), HL-60 tumor bank. 2794

Downloaded from cancerres.aacrjournals.org on July 12, 2011 Copyright © 1995 American Association for Cancer Research

AG17 INHIBITSTUMOR GROWTHBY MITOCHONDRIALIMPAIRMENT

H3C@ / C

CH3

/@ CH3

RESULTS

Cell Growth and Macromolecular Synthesis. The MTI' colori

CN

‘@%%@

I

metric assay for viable cell mass was used to determine the effect of “continuous― drug exposure (6 day) on cell growth. AG17 inhibited the growth of 13 human tumor cell lines (leukemia, ovarian, brain, colon, breast, melanoma, renal, and lung carcinoma cell lines, Table 1) examined, with GI50 values ranging from 0.7 to 4.0 @LM. The compound was found to be relatively equipotent in causing GI50 in these cell lines after 6-day exposure, but differentially sensitive in achieving total growth inhibition. The drug concentration required for

CN

H3C—C—--CH3 CH3 Fig. 1. NSC 242557 or AG17.

TGI was higher in the glioblastoma cell line SNB-19, the breast The viable cell mass in each plate was measuredby MIT reduction as described above.

Macromolecular Synthesis. HL-60(TB) leukemia cells (5000/well) were

carcinoma line MCF-7, and the renal carcinoma cell line A498 (P < 0.05, Table 1). For all of the other tumor cell lines, steep concentration-effect curves for growth inhibition were observed with

GI@@ and TGI values spanning a narrow range (1—[email protected]), as shown in AG17for 2, 4, 8, and24 h, andpulsedwith0.5 pCi/1Opd/well[3H]thymidine Fig. 2 for HCT-116, Cob 205, HL-60(TB), and K-652 cell lines. plated in a 96-well plate in 100 p@lmedium. After 24 h cells were exposed to (specific activity, 85 Ci/mmol; Amersham Life Science, Arlington Heights, IL)

to measure DNA synthesis, [3H]leucine(specific activity, 153 Ci/mmol; Am ersham) to measure protein synthesis, or [3H]uridine (specific activity, 46

Ci/mmol;Amersham)to measureRNA synthesisfor the final 2 h of drug exposure. Cells were harvested on a filter mat by a 96-well cell harvester

(Skatron, Inc., Sterling, VA). Radioactivity on the filters was measured using a Packard 150Q Tri-Carb Liquid Scintillation Analyzer (Packard, Meriden, CI). Effects of AG17 on ATP Content The mass of ATP present in a 5% trichloroacetic acid cell extract was determined with the firefly luciferinlucif erase system (17). FIL-60(TB) cells (2 X 106)were plated into 6-well plates containing 4 ml medium/well. After 24 h, drugs were added for the indicated period, and then cells were transferred to tubes, washed twice with ice-cold PBS, and lysed by addition of 1 ml ice-cold 5% trichioroacetic acid. Ten pi AlP standard or trichloroacetic acid extract were mixed with 900 gxl ThM buffer (100 mM Tris-acetate pH 7.7, 15 mti MgSO4, and 2 mM EDTA), and

light output was measured for 20 s at 25°Cin a Monolight 2010 biolumines cence photometer after injection of 100 .dluciferin and/or luciferase according to instructions from the manufacturer (Analytical Luminescence Laboratory, San Diego, CA). The AlP standard calibration curve was tested for an effect of addition of the drug chromophore, but no change in luminescence was observed. Triplicate measurements for each point were taken, and experiments

were repeatedthree times.The drug effectwas quantitatedin pmolfrom the arbitrary luminescence

units of the standard curve. Protein precipitated from

the extract was solubilized and measured using the Pierce BCA protein assay (Pierce, Rockford,

IL). Results were therefore expressed

as pmol AlT/mg

protein. Effects of AG17 on Rhodamine 123 Retention. The fluorescent stain rhodamine 123 (purchased from Eastman Kodak Co., Rochester, NY) was used as a probe to assess the mitochondrial membrane potential of living HL.60(FB) tumor cells (18—20).Cells were plated in a 96-well plate at a cell density of 20,000 cells/well in 100 pi medium and cultured for 24 h. Cells were prestained for 10 mm at 37°Cafter addition of the dye to a final

To assess the “reversibility― of drug effect, cells were exposed for varying time periods to concentrations (Table 1) which just inhibited cell growth completely (TGI) after 6 days of continuous exposure to drug. Drug was then removed at various intervals, and cells were incubated in drug-free medium for a total of 6 days. Table 2 shows that HL-60(TB) was the most sensitive cell line, since only 12 h of exposure was needed to effect persistent complete inhibition of growth, whereas K-562 and SK-MEL-5 required 48 h and other cell lines (Cob 205, HCT-116, and HOP-92) needed up to 72 h of drug exposure to achieve irreversible inhibition of tumor cell growth when assayed by viable cell mass 6 days after drug removal.

In an effort to delineate the biochemical basis for the antiprolifera tive activity of AG17, we studied the drug effect on macromolecular synthesis (Fig. 3). After only 2 h of exposure to the TGI concentra tions (1.5 @.LM), synthesis of protein, RNA, and DNA were inhibited by 50% in HL-60(TB) cells. Eight h of drug exposure caused an approx imate 90% inhibition of thymidine and uridine incorporation and 70% inhibition of leucine incorporation in these cells. There was no pref erential inhibition of macromolecular synthesis, as AG17 inhibited DNA, RNA, and protein synthesis by 50% at approximately 1.5 ,.@M. AlP Content. Since AG17 had been reported to inhibit tyrosine kinases in vitro (5, 10), we examined the effect of the drug on total phosphorylated protein content in living HL-60(TB) cells. We found that exposure to AG17 at 2 p.M did not differentially affect 32PO4 AGJ7―GI5@TGICell Table 1 Inhibition of tumor cell growthby SELeukemiaK-5621.10±0.152.00± line(@xM)

0.07HL-60(TB)0.70 0.11Molt-41.00

concentrationof 10 gxg/mI/well.Excess dye was removedby washingthe

0.12SR2.50 0.10OvarianOvcar-31.55

±

±0.14150 ±0.053.95

±

±0.204.80

± ±

0.36BrainSNB..19b1.80

±0.152.40

±

1.50ColonCob

±0.0939.00

±

Cell-associatedfluorescencewas quantitatedin fluorescentlight units using

0.19HCT-l161.75±0.034.15 2051.90 ±0.13BreastHS

±0.182.90

±

the Cytofluor 2300 plate reader. This staining procedure, excluding the final cell lysis step, was also used to prepare cells for fluorescence microscopic examination.

0.3MCF-7―4.00 578T1.20 0.29Melanoma5K-MEL-S1.50

±0.092.15

±0.2216.00

± ±

0.47RenalA498―1.75

±0.254.80

±

0.34LungHOP-fl2.00

±0.0310.50

±

plates twice with PBS followed by centrifugation at 1200 rpm for 10 min and discarding the supernatant by blotting them carefully onto paper towels. The stained HL-6oçrB) cells were resuspended in 200 @.d of drug-free medium (cell control) as well as medium containing drug (0.01—100 gxM). Rodamine 123 release from mitochondria was measured at 0, 1, 2, and 4 h. Cells were washed twice with PBS as described before and lysed in 100 @xl/welldistilled water.

Electron Microscopy. HL-60(TB) cells were treated with AG17 for 2 to 24 h, and iO@cells were then washed twice with PBS and fixed in 1.25% glutaraldehyde. Cell preparation for electron microscopic ultrastructural stud

ies was performed as described previously (21) using a Hitachi H-7000 electron @

±SE(tiM)

±0.096.30

microscope.

Every experiment described in “Materials and Methods― was performed as three independent determinations, each with at least three replicate values.

a Values

are

based

on

a 6-day

MIT

determinations. b

< o.os.

2795

Downloaded from cancerres.aacrjournals.org on July 12, 2011 Copyright © 1995 American Association for Cancer Research

assay

and

±0.44 represent

the

mean

of three

AG17 INHIBITS TUMOR GROWTh BY MITOCHONDRIAL IMPAIRMENT

Rhodamine 123 Retention. The decrease in AlP content could reflect a direct effect of the drug on mitochondrial functions in living cells. Since loss of previously internalized rhodamine 123 indicates disruption of mitochondrial membrane potential, we studied rhodamine 123 retention after AG17 exposure in HL.60(TB) cells. Rhodamine 123 is known to insert into mitochondria as a function of the negative electrochemical gradient across the mitochondrial mem brane, and its release from prestained cells is a recognized action of compounds which are respiratory poisons (18—20).Fig. 5A demon strates that AG17 interferes with rhodamine 123 retention after only 1 h of exposure to drug, and was more potent than known intracellular inhibitors of mitochondrial function (antimycin A) or uncouplers of oxidative phosphorylation (oligomycin and carbonyl cyanide m-chlo rophenylhydrazone, respectively; Fig. 5B and Refs. 18 and 19). Fig. 6

100-

@80C

0

0

,@

60

0

@

ci 20

further underscores the obvious loss of rhodamine 123 staining after

C 0.1

1

10 100

only 1 h of exposure to 2 p@M AG17, using fluorescence microscopy. Effect of AG17 on Mitochondrial Ultrastructure. Direct exam ination of the mitochondrial membranes of HL-60(TB) cells by dcc tron microscopy revealed a loss of mitochondrial membrane integrity after 2 h of exposure to 2 ,.LMAG17 (Fig. 7B), as compared to

1000

AG17(pM) Fig. 2. Concentration-effect

plot of AG17 for cell growth. Exponentially growing cells

received the indicated concentrations of AGI7 for 6 days prior to the addition of MTI.

@

The results plotted are the mean ±SD (n = 6) percentage absorbance at 540 nm compared to vehicle-treated control cells (C). HCT-1 16 (, 100% 0.84), Cob 205

120

B

(•, 100% = 0.76), HL-60(TB) (A,100% = 1.49),andK-562 ( , 100% = 1.37).

100 80

Table 2 Reversibility of drug effectExposure toCell

60

time

40

lineachieve TGIK-56248

20

hHL-60(TB)12 hCob 20572 daysHC'T-11672 hSK-MEL-548 hA49872 daysHOP-9272

0

h—6

C 0.01 0.1 1 10 AGI7 (pM)

C 0.01 0.1 1 A017(pM)

10

C 0.01 0.1 1

10

A017(pM)

Fig. 3. Time course of AG17 effect on macromolecular synthesis. HL-60('FB)cultures

h—6

were exposed for 2 h (U), 4 h(I), and 8 h (A)and pulsed for the final 2 h of drug exposure with [3Hlleucine (A, 100% at 2, 4, and 8 h 220, 241, and 255 cpm), l3Hluridine (B,

h

100% at 2, 4, and 8 h

584, 743, and 1350 cpm), and I3HJthymidinc(C, 100% at 2, 4,

and 8 h = 896, 1182, and 3081 cpm). Results are shown as percentage of vehicle control (C) as mean ±SD (n = 6).

incorporation into cellular proteins but led to a global decrease in phosphorylation 6—24h after treatment. Western blotting of whole cell lysates with antiphosphotyrosine antibodies after immunoprecipi tation of phosphotyrosine-containing proteins did not suggest a spe cific decrease in the phosphotyrosine mass of drug-exposed cells

(2 p@M up to 24 h, datanot shown).Theseresults,aswell asthe early nonspecific inhibition of DNA, RNA, and protein synthesis (Fig. 3) raised the possibility that AG17 may rather have an impact on met abolic processes central to all three macromolecular synthetic path ways. At this point we also became aware through searching a chemical structure data base that AG17 is identical to SF 6847, a potent uncoupler of oxidative phosphorylation (12). We therefore monitored

Al?

mass over a time course of 24 h and compared

cells

(Fig.

4). After

only

2 h of exposure,

AG17

I-

a. Dl

E

a.

AG17

to antimycin A, a known inhibitor of oxidative phosphorylation living

C

in

(2 p.M)

0

E

decreased the AlT content in HL-60(TB) cells by 50%, as compared 0. to untreated control, whereas antimycin A (10 @tM), a well-recognized mitochondrial poison, decreased the Al? content by 25%. After 24 h of exposure, ATP levels were 15% of control in AG17-treated cells as compared to 45% of control in antimycin A-treated cells. This rapid decrease of ATP content at drug concentrations causing TGI in HL-60(TB) cells clearly suggests that the disruption of cellular 12 energy balance by AG17 must be considered as a possible mode of lime (h) action for subsequent growth inhibition of tumor cells, irrespective Fig. 4. Effect of AG17 on ATP content. HL-60(TB) cells were exposed to 2 @iM AG17 of the effects of the drug on PTK activity. Qualitatively similar (@)for2,6,12,and24handto10ps@i antimycin A(U)for2and24h.TheAlP content results were obtained in K562 chronic myelogenous leukemia cells in pmol/mg protein was assayed by luciferin/luciferase reaction. Also plotted are untreated (R) and vehicle control (0) HL-60(TB) cells. Results are shown as mean ±SD (n 3). (data not shown). 2796

Downloaded from cancerres.aacrjournals.org on July 12, 2011 Copyright © 1995 American Association for Cancer Research

AGI7 INHIBITSTUMOR GROWTHBY MITOCHONDRIALIMPAIRMENT

phosphotyrosine containing proteins of HL-60(TB) cells analyzed by Western blotting was not detectable even after 24 h of exposure to AG17. Earlier in vitro investigations with AG17, designated as SF 6847, had shown that AG17 could act in purified preparations of mitochon dna as an uncoupler observation therefore

I

@

energy

, 0.1

C 0.01

-9 1

.. 10

100

metabolism

A

I

sis (DNA,

0.1

1

10

100

DrugConcentration (pM) Fig. 5. Rhodamine 123 retention after exposure to AG17. A, mitochondria membrane status in HL-60çrB)cells was assessed by measuring the fraction of rhodamine 123 retained in drug-treated

as compared

to vehicle-exposed

could be the basis for its antiproliferative

activity.

Indeed, as demonstrated in Figs. 4 through 7, significantly decreased Al? levels and loss of rhodamnine 123 staining caused by mitochon drial injury is an early effect in living cells at concentrations near the G150 and TO! in multiple tumor cell types (Tables 1 and 2). Considering the obvious central role that ATP plays in maintaining cellular anabolic functions, a significant drop of ATP concentra tion would then lead to a variety of other perturbations, consistent with our findings of an early inhibition of macromolecular synthe

AG17(pM)

C 0.01

of oxidative phosphorylation (12, 13). This suggested to us that perturbation of cellular

cells (C). The fraction of

fluorescent light units remaining after prestaining with rhodamine 123 and exposure to the indicated concentrations of AG17 at time 0 (0, 100% = 1538), 1 h (0, 100% = 953), 2

RNA,

protein;

Fig.

3), but

also

with

other

reported

effects of the drug such as inhibition of EGF receptor phosphoryl ation, IL-2-induced proliferation, or stimulation of glucose uptake (5, 9, 11). Therefore, reports of the effects of AG17 on signal transduction by PTK inhibition (e.g., Ref. 5) must be tempered with the caveat that AG17 can induce ATP depletion following mitochondrial injury. Our results emphasize that studies with other tyrphostin-like com pounds (e.g., Refs. 2, 4, 6, 10, and 22) in living cells must individually address whether their effects do only derive from inhibition of signal transduction, and not from other effects on cellular metabolism. While

some tyrphostins can definitively act as very specific inhibitors of signal transduction by tyrosine protein kinases (6, 7), each tyrphostin oligomycin(G, 100%= 902),antimycinA (Li,100%= 1000),and carbonylcyanide should be examined critically for non-PTK-related effects before it is m.chlorophenylhydrazone (Y, 100% = 890) for 4 h. used as a tool to dissect these pathways. In the case of AG17, the effect of the drug on PDGF-mediated proliferation in vascular endo thelial cells occurs at a concentration with OI@, (0.04 p.M) as com vehicle-treated control cells (Fig. 7A). The plasma cell membrane pared to serum-stimulated proliferation (GI5@,1.23 p.M; Ref. 9). Thus, it is possible that at very low concentrations of drug, specific inhibi remained intact in both cases. After 24 h of treatment, barely discern tion of kinase-mediated signaling will predominate over the effects on ible contours of former mitochondna were visible (Fig. 7C). cellular AlT production. It is clear that this possibility must be established for each compound of the malononitrile family of tyr DISCUSSION phostins (1, 4, 6) and potentially each cell type in which these agents The experiments presented in this article demonstrate that AG17 are used. inhibits the growth of a variety of human tumor cell lines with G150 Although mitochondria can be considered a cellular target for ranging from 0.7 to 4 p.Min a 6-day growth inhibition assay; cell lines AG17, the precise nature and mechanism for its mitochondrion differ in their susceptibility to irreversible growth inhibition, requiring directed toxicity remains unknown. Young et a!. (23) have also exposure from 12 h for HL-60(TB) to >72 h for Cob 205 and A498. demonstrated damaging effects of tyrosine kinase antagonists includ AG17 indiscriminately inhibits DNA, RNA, and protein synthesis ing quercetin, genistein, and certain tyrphostin-like compounds on shortly after addition to HL-60(TB) cells. AlT levels decline, reflect mitochondrial function in isolated mitochondria and isolated fat cells. ing damage to mitochondria at drug concentrations where inhibition However, those studies used concentrations of inhibitors at 100—200 of macromolecular synthesis is evident after only 2 h of exposure to p@M,yet our studies demonstrate deleterious effects in living HL-60 the drug (Fig. 3). cells at 2 @.LM and lower, at concentrations similar to those producing Previous studies had focused on the capacity of AG17 to inhibit inhibition of cell growth. EGF and PDGF receptor tyrosine protein kinases using in vitro The fact that different tumor cell lines require different periods of reactions or in cell cultures stimulated with EGF (5) or PDGF (1, 9). drug exposure to achieve an irreversible effect on cell growth The drug had also been characterized as a potent inhibitor of a variety (Table 2) is of interest and could be explained by differences in Al? of functions in lectin and IL-2-stimulated human peripheral mononu “demand― by the cells to sustain viability or differences in the rate of clear cells (1 1). However, our current studies suggest that the action mitochondrial turnover or assembly. Additional studies must address of AG17 at concentrations which inhibit the growth of cultured tumor how the drug may differentially affect mitochondrial structure or cell lines may not be due to specific inhibition of protein kinases. function in “sensitive― as compared to “resistant― cell types. Although Indeed, extensive efforts to demonstrate inhibition of breast carci “classic― uncouplers of electron transport are not expected to be useful noma cell growth in a manner that correlated with inhibition of agents in preclinical studies owing to expectation of low therapeutic specific tyrosine kinases and at concentrations where Al? levels were index, it is conceivable that sensitization to currently used antitumor preserved were not successful.4 Furthermore, a specific decrease in agents may occur after a decrease of Al? in sensitive cell types and may be a basis for further further development of AG17. The exper h (t@.,100% = 1694) and 4 h (0,

100%= 978) is depicted as mean ±SD (n = 3). B,

rhodamine123retention(in% fluorescentlightunits)by HL-60(TB)cellsexposedto the indicated concentration ofAGl7 for 0 h (0, 100% = 1527) or ofAGl7 (0, 100% = 945),

4 B.

Sausville

and

0.

Kaur,

unpublished

data.

iments

presented

here,

however,

2797

Downloaded from cancerres.aacrjournals.org on July 12, 2011 Copyright © 1995 American Association for Cancer Research

should

be considered

carefully

in

AGI7 INHIBITSTUMORGROWTh BY MITOCHONDRIALIMPAIRMENT

ø@,.

I

B

a

‘a Fig. 6. Fluorescence photomicrographs of rho damine 123-stained HL-60(TB) cells. A, bright stained vehicle control cells 1 h after treatment. B,

@

significantlydiminishedfluorescenceafter A617 (2 psi) exposure in the same time period. X 320.

C. * ? ‘I,

y

•

Et •

so

C

Fig. 7. Mitochondrial membrane damage by AG17. In A, HL-60(TB) cells were exposed to DMSO. In B, cells were exposed to 2 p.MAG17 for 2 h. In C, cells were exposed to AG17 for 24 h. Damage to the membrane of several mitochondria (m) is evident, as indicated by arrows. The 24-h view presented is of a representative viable cell; at that time, many

cells were greatly altered in morphology. Bars, 1 @tm. 2798

Downloaded from cancerres.aacrjournals.org on July 12, 2011 Copyright © 1995 American Association for Cancer Research

AG17 INHiBITSTUMOR GROV.THBY MITOCHONDRIALIMPAIRMENT

planning the further use of the agent as a modulator of protein tyrosine kinase-dependent signal transduction pathways.

11. Lander, H. N., Levine, D. M., and Novogrodsky, A. Agonistic effects of tyrphostins

on human peripheral mononuclear cells. Cell. Immunol., 144: 155—168, 1992. 12. Freisleben, H. J., Fuchs, J., Mainka, L., and Zimmer, 0. Reactivity of mitochondrial sulfhydryl

groups

cm-inhibited

ACKNOWLEDGMENTS

toward

dithionitrobenzoic

and uncoupling

conditions.

acid and bromobimanes

under

oligomy

Arch. Biochem. Biophys., 266: 89—97,

1988.

We thankKunioNagashima,who performedtheelectronmicroscopicwork, Ruth Herringfor consistantpreparationof the tumorcell lines, and Matthew Gonda for his help in evaluating electron micrographs.

13. Terada, H., Kumazawa, N., Ju-Ichi, M., and Yoshikawa, K. Molecular basis of the protonophoric and uncoupling activities of the potent uncoupler SF-6847 ((3,5di-tert-butyl-4-hydroxybenzylidene)malononitrile) and derivatives. Regulation of

their electronic structures by restricted intramolecular rotation. Biochim. Biophys. Acta, 767: 192—199,1984. 14. Alley, M. C., Scudiero, D. A., Monks, A., Hursey, M. L., Czerwinski, M. J., Fine, D. L., Abbott, B. J., Mayo, J. G., Shoemaker, R. H., and Boyd, M. R. Feasibility of

REFERENCES

drug screening

1. Gui@ A., Yaish, P., Gilon, C., and Levitzki, A. Tyrphostins 1: synthesis and

biological activity of protein tyrosine kinase inhibitors. J. Med. Chem., 32: 2344-2362, 1989.

with panels

of human

tumor cell lines using a microculture

tetrazolium

assay. Cancer Res., 48: 589—601,1988. 15. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application

to proliferation and cytotoxicity assays. J. Immunol. Methods, 65: 55—63,1983. 16. Monks, A., Scudiero, D., Skehan, P., Shoemaker, R., Paull, K., Vistica, D., Hose, C.,

2. LeVitZki, A. Tyrphostins—potential antiproliferative agents and novel molecular tools. Biochem. Pharmacol., 40: 913—918,1990. 3. LCVItZki,A. Tyrphostins: tyrosine kinase blockers as novel antiproliferative agents

Langley, J., Cronise, P., Vaigro-wolif, A., Gray-Goodrich, M., Campbell, H., Mayo, J., and Boyd, M. Feasibility of a high-flux anticancer drug screen using a diverse

anddissectoisof signaltransduction.FASEBJ., 6: 3275—3282, 1992.

panel of cultured human tumor cell lines. J. Natl. Cancer Inst., 83: 757—766,1991.

4. Levitzki, A., Omit, A., Osherov, N., Posner, I., and Gion, C. Inhibition of protein

17. DeLuca, M., and McElroy, W. D. Purification and properties of firefly luciferase. Methods Enzymol., 57: 3—15,1978. 18. Johnson, L. V., Walsh, M. L., Bockus, B. J., and Chen, L. B. Monitoring of relative

tyrosinc kinases by tyrphostins. Methods Enzymol., 201: 347—361,1991. 5. GIllespie, J., Dye, J. F., Schachter, M., and Guillou, P. J. Inhibition of pancreatic cancer cell growth in vitro by the tyrphostin group of tyrosine kinase inhibitors. Br. I. Cancer, 68: 1122—1126,1993. 6. Anafi, M., Gazit, A., Gion, C., Ben-Neriah, Y., and Levitzki, A. Selective interac

mitochondrial membrane potential in living cells by fluorescence microscopy. J. Cell Biol., 88: 526—535,1981.

tions of transforming and normal abi proteins with, AlT tyrosine-copolymer substrates and tyrphostins. J. Biol. Chem., 267: 4518—4523, 1992. 7. Knit, G., Gazit, A@,LeVitZki,A., Stowe, E., Cooney, D. A., and Sausville, E. Tyrphostin

inducedgrowthinhilrition: correlationwitheffecton p210@ autokinaseacitivityin 1362 chronicmyelogenous leukemia.AnticancerDrugs,5: 213-222,1994. 8. Supko, J. 0., Malspeis, L., Sausville, E. A., Burger, A., Alley, M., and Grever, M. R. Identification of the antitumor activity and preclinical evaluation of the benzyliden emalononitrile derivative NSC 242557. Proc. Am. Assoc. Cancer Rca., 35: 424, 1994. 9. Bilder, G. E., Krawiec, J. A., McVety, K, Omit, A., Gion, C., Lyall, R., Zilberstein,

A., Levitzki, A., Perrone, M. H., and Schreiber, A. Tyrphostins inhibit PDGF-induced

DNAsynthesisandassociatedearlyeventsin smoothmusclecells.Am.J. Physiol., 260: C721-C730, 1991. 10. LeVitZki, A., and Gion, C. Tyrphostins as molecular tools and potential antiprolif

erative drugs. Trends Pharmacol. Sci., 12: 171—173, 1991.

19. Rago, R. P., Brazy, P. C., and Wilding, G. Disruption of mitochondrial function by suramin measured by rhodamine 123 retention and oxygen consumption in intact DU145 prostate carcinoma cells. Cancer Res., 52: 6953—6955, 1992. 20. Rago, R. P., Mitchen, J., Cheng, A. L., Oberly, T., and Wilding, 0. Disruption of

cellular energy balance by suramin in intact human prostatic carcinoma cells, a likely antiproliferative mechanism. Cancer Res., 51: 6629—6635, 1991. 21. Evermann, J. F., Heeney, J. L., McKeirnan, A. J., and O'Brian, S. J. Comparative

features of a coronavirus isolated from a cheetah with feline infectious peritonitis. Virus Res., 13: 15—28, 1989. 22. Lamb, D. J., and Sankararaman, S. Tyrphostins inhibit sertoli cell-secreted growth factor stimulation of A431 cell growth. Recent Prog. Horm. Res., 48: 511—516, 1993. 23. Young, S. W., Poole, R. C., Hudson, A. T., Halestrap, A. P., Denton, R. M., and Tavare, J. M. Effects of tyrosine kinase inhibitors on protein-kinase independent systems. FEBS Left., 316: 278—282, 1993.

2799

Downloaded from cancerres.aacrjournals.org on July 12, 2011 Copyright © 1995 American Association for Cancer Research