Chemical modulation of bleomycin induced toxicity

ht. J. Radiation Oncology Biol. Phys.. Vol. 12, pp. 1367-I 370 Printed in the U.S.A. All rights reserved.

Copyright

0

0360-3016/86 1986 Pergamon

$3.00 + .oO Journals Ltd.

??Session V

CHEMICAL MODULATION

OF BLEOMYCIN

KATHERINE A. KENNEDY, PH.D. ‘, WILLIAM N. HAIT, M.D.,

INDUCED

TOXICITY

PH.D.~ AND JOHN S. LAZO, PH.D.~

‘Department of Pharmacology, The George Washington University 2300 I Street NW, Washington, DC 20037; and Departments of 2Medical Oncology and 3Pharmacology Yale University, P. 0. Box 3333, New Haven, CT 065 10 Both lidocahre (LIDO) and the caImoduIIn(CaM) antagonists, pimozlde (PIM) and trlfhroperazhre0, enhanced bleomycin (BLM) induced cytotoxicity and DNA damage. The toxicity with the combination of BLM and CaM antagonists represented true pharmacological synergism and was observed with the addition of the CaM antagonist either during or after BLM exposure. Additionally, the DNA damage of BLM and the BLM-like drugs, talisomycin Sloa (TAL) or peplomycin (PEPLO), was also enhanced by CaM antagonists. LIDO, which similarly increased the lethal effects and DNA damage of BLM in L1210 cells, was also effective only during or after BLM exposure. The data presented here indicate that the modulation of toxicity seen with these drug combinations is reflected by changes in DNA integrity. Furthermore, these data suggest that the inhibition of DNA repair processes may be at least partially responsible for the enhanced toxicity and DNA damage when CaM antagonists or LID0 are added to BLM. Bleomycin, Calmodulin antagonists,

Alkaline elution, Toxicity, L1210 cells, Lidocaine.

INTRODUCTION

serum as previously described.3 For experiments in which the potentiation of BLM toxicity by CaM antagonists or LID0 was investigated, cells were seeded at 7 X 103/ml, drugs were added, and growth inhibition was assessed 72 hr after drug addition. The data were analyzed for potentiation or simple additivity by isobologram analysis as previously described. ’

Bleomycin (BLM), an effective antitumor agent, is thought to cause cytotoxicity through its ability to induce single and double strand breaks in DNA. BLM-induced DNA damage and cytotoxicity can be modified by treating cells with the local anesthetic, lidocaine (LIDO), as previously shown,3,5*6or with several calmodulin (CaM) antagonists, including pimozide (PIM).4 To determine if the damage and toxicity observed previously3-5 were specific for BLM, the effects of LID0 and the CaM inhibitors, PIM and trifluoperazine (TFP), on the toxicity and DNA damage induced by BLM and the second generation BLM analogs, talisomycin Slob (TAL) and peplomycin (PEP) were investigated.

DNA damage DNA damage was assessed by alkaline elution techniques as previously described.3,4 RESULTS In previous studies,4 the cytotoxicity of BLM towards cultured L 12 10 tumor cells was enhanced upon the concurrent treatment of the cells with CaM inhibitors (including PIM and TFP) and BLM. Factors such as reduced inactivation of BLM, or elevated cellular BLM content were not responsible for the enhanced cytotoxicity of the

METHODS Cells Experiments were performed using L 12 10 cells grown in suspension in Fisher’s medium* containing 10% horse

Presented at the Chemical Modifiers for Cancer Treatment Conference, Clear-water,FL, 20-24 October 1985. Reprint requests to: Dr. Katherine A. Kennedy, Department of Pharmacology, The George Washington University, 2300 I Street NW, Washington, DC 20037. Acknowledgements-This research was supported in part by American Cancer Society grants CH- 174 (JSL), CH-274 (KAK),

and CH-302 (WNH), a Swebilius award (WNH), USPHS grant CA-36552 (WNI-I), an NIH Research Career Development Award CA-O1012 (JSL), and a Young Investigator Award from Eli Lilly and Co. (KAK). Accepted for publication 25 February 1986. * GIBCO, Grand Island, NY.

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l% +

CONTROL

d IL

1OlrM

TFP

.

August 1986, Volume 12, Number 8 Table 1. Enhanced DNA damage by BLM and its analogs in the presence of CaM antagonists or lidocaine

A

g

Treatment

% drug-induced damage*

ii a 5 i 0.4b w

BLM BLM + PIM BLM + TFP BLM + LID0

100 190 219 281

TAL TAL + PIM TAL + TFP TAL + LID0

100 173 154 423

PEPLO PEPLO + PIM PEPLO + LID0

100 213 490

I

1 .o

0:S FRACTION

0%

0:4

INTERNAL

0:2

STANDARD

DNA ON

FILTER

Fig. 1. Enhancement of BLM-induced DNA damage by TFP. L12 10 cells were incubated for 1 hr with 10 MM TFP (A), 25 PM BLM (O), or the combination, 10 PM TFP and 25 PM BLM (0). Immediately after treatment, cells were assayed for DNA damage as described in the Methods section.

combination.4 The amount of DNA damage as measured by alkaline elution analysis, however, was increased in cells treated with the combination of PIM and BLM over that observed in cells treated with BLM alone.4 The enhancement of BLM-induced DNA damage is not restricted to PIM, but is shared by another structurally distinct CaM antagonist as shown in Figure 1. In previous studies, as well as the present investigation, both PIM and TFP were used at concentrations that did not result in any measurable DNA damage or decrement in colony forming ability. Figure 2 indicates the isobologram analysis for the interaction of PIM and TFP with BLM. The data show that

0 BLM 0 BLM

20

40 PERCENT

60 lC50

* Data are expressed as % of the rad equivalent DNA damage induced by treating L12 10 cells with BLM or TAL or PEPLO alone and represent the means of 2-3 determinations.

for either CaM antagonist, pharmacological synergism with BLM was observed. Table 1 indicates that the DNA damage induced by BLM, TAL, or PEPLO was increased in cells coincubated with PIM or TFP. These data further suggest that the enhanced toxicity and DNA damage observed when BLM-like drugs are combined with CaM antagonists are not specific to either a particular CaM antagonist or to a specific BLM-like drug. The effects of PIM on BLM treated cells when PIM was added either concurrently with BLM or after BLM

A2 + PIM A2 + TFP

60 BLM

100 A2

Fig. 2. Synergistic interactions between BLM and CaM antagonists. Isobole for L12 10 cell growth inhibition with combinations of PIM (0) or TFP (0) with BLM. Cells were treated continuously with either each drug alone or with various combinations of CaM antagonists and BLM. Three days after incubation, the total number of cells was determined. Similar results have been obtained with TAL and TFP used in combination.

Fig. 3. Effect of PIM on the repair of BLM-induced DNA damage. L12 10 cells were incubated with 50 PM BLM (0) for I hr. 50 PM BLM for 1 hr followed by 1 hr incubation in drug free medium (B), 50 PM BLM for 1 hr followed by a 1 hr incubation in medium containing 5 MM PIM only (Cl), or with the combination of 50 PM BLM and 5 PM PIM for 1 hr (0). After treatment, the extent of DNA damage was assayed by alkaline elution. Results from a typical experiment are shown.

Bleomycin induced toxicity 0 K. A.

treatment are shown in Figure 3. The combination of BLM and PIM, produced enhanced DNA damage when compared to cells treated with BLM alone. If cells were treated with BLM and then incubated for 1 hr in drugfree medium, approximately 25% of the drug induced damage was repaired (Figure 3 and Table 2). In contrast, L 12 10 cells treated for 1 hr with BLM followed by incubation for 1 hr in fresh medium containing only PIM, showed DNA damage that was equivalent to that found in cells treated concurrently with BLM and PIM for 1 hr. Previous reports3 have shown that LIDO, a local anesthetic, also enhances the effects of BLM in L 12 10 cells. The data shown in Figure 4 and Table 2 support these conclusions. Enhanced toxicity3,5,6and DNA damage was dependent upon the sequence of drug addition. Enhanced toxicity was found only under those conditions that an increase in DNA damage was observed2 (Figure 4 and Table 2). DISCUSSION Both LID0 and the CaM antagonists, PIM and TFP, enhance BLM induced cytotoxicity and DNA damage. This enhancement in toxicity was not specific for either the CaM antagonist used4 or for the particular BLM analog employed. The CaM enhancement of BLM toxicity represented true pharmacological synergism (Fig. 2) and required the addition of the CaM antagonist either during

Table 2. The effects of LID0 and PIM on the repair of BLM-induced DNA damage

Treatment*

% of BLM-induced DNA damage?

Enhancement of BLM toxicity

BLM BLM: 1 hr repair

100 72

No

BLM + PIM BLM: 1 hr repair + PIM

162

Yes

209

Yes

225

Yes

358 85

Yes No

BLM + LID0 BLM: 1 hr repair + LID0 LIDO: BLM

* L12 10 cells were treated with BLM for 1 hr or allowed to repair the damage in BLM-free medium containing either vehicle or PIM or LIDO. In addition, L 12 10 cells were pretreated for 1 hr with LIDO, washed free of drug, and then incubated with BLM for 1 hr (LID0 BLM). After treatment, the cells were assayed for DNA damage using alkaline elution methods. t Data are expressed as the % of the rad equivalent DNA damage produced by treating cells with BLM alone for 1 hr. DNA damage data represent the mean of 2-3 experiments. Lethality was measured by colony formation and has been reported elsewhere.*y3

KENNEDY

et al.

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Fig. 4. Effect of LID0 on BLM-induced DNA damage. L1210 cells were incubated with 25 PM BLM for 1 hr (A); 25 PM BLM for 1 hr followed by a 1 hr incubation in BLM-free medium containing 8 mM LID0 (A); 8 mM LID0 for 1 hr (a); 8 mM LID0 for 1 hr, followed by a 1 hr incubation in fresh medium, and then a subsequent 1 hr incubation with 25 PM BLM (0); or a 1 hr incubation with the combination of 25 pM BLM and 8 mM LID0 (W). After treatment, the extent of DNA damage was assayed by alkaline elution analysis.

or after BLM exposure. Other investigators2 have suggested that inhibition of DNA repair processes was an important component in the enhancement of BLM toxicity by CaM antagonists. Our data lend additional support to the possibility that CaM antagonists may produce at least part of their effects through the inhibition of repair of BLM-induced damage. LID0 is an example of another agent that can potentiate the actions of BLM in L12 10 cells.3,5,6Other investigators have also shown that local anesthetics, such as LID0 and dibucaine, can enhance the cytotoxic and DNA damaging potential of BLM in cultured cells. The data presented here indicate that the sequence specificity for toxicity3 also holds for the enhancement of DNA damage. This sequence selectivity may also indicate an effect of LID0 on repair processes for BLM-induced damage. Although the inhibition of DNA repair may be responsible for some of the effects of CaM antagonists or LID0 on BLM toxicity, CaM antagonists do not affect the toxicity of other agents which induce repairable DNA lesions.3,4 Similarly, LID0 potentiation of BLM induced toxicity is fairly specific for BLM-like drugs and does not occur with other agents such as etoposide or mitomycin C.3*4In addition, CaM antagonists apparently do not affect potentially lethal damage produced by X-irradiation.7 Furthermore, the extent of repair by L12 10 cells of BLM induced damage was not extensive. Thus, inhibition of repair by CaM antagonists or LID0 may represent only a small component of the synergistic activity observed for combinations of BLM with these agents. Additional factors such as altered intracellular drug distribution may be involved.

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REFERENCES 1. Berenbaum, M.C.: Criteria for analyzing interactions between biologically active agents. Advances in Cancer Res. 35: 269, 1981. 2. Chafouleas, J.G., Bolton, W.E., Means, A.R.: Potentiation of bleomycin lethality by anticalmodulin drugs: A role for calmodulin in DNA repair. Science 224: 1346, 1984. 3. Laze, J.S., Braun, I.D., Meandzija, B., Kennedy, K.A., Pham, E.T., Smaldone, L.F.: Lidocaine potentiation of bleomycin A2 and DNA strand breakage in L 12 10 and human A-253 cells. Cancer Res. 45: 2103, 1985. 4. Lazo, J.S., Hait, W.N., Kennedy, K.A., Braun, I.D., Meandzija, B.: Enhanced bleomycin-induced DNA damage and cytotoxicity with calmodulin antagonists. Mol. Pharmacol. 27: 387, 1985.

5. Mizuno, S., Ishida, A.: Selective enhancement of bleomycin cytotoxicity in local anesthetics. Biochem. Biophys. Res. Commun. 105: 425, 1982. 6. Misuno, S., I&da, A.: Selective enhancement of the cytotoxicity of the bleomycin derivative, peplomycin, by local anesthetics alone and combined with hyperthermia. Cancer Res. 42: 4726, 1982. 7. &linger, D.N., Loh, S., Dethlefsen, L.A.: A role for calmodulin in the radiation response of proliferating and quiescent 67 murine mammary carcinoma cells. 33rd Annual Meeting Abstracts, Radiation Research Society, Ei-8. 1985, pg. 48.