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Pro-apoptotic interactions between XK469 and the peripheral benzodiazepine receptor
Cancer Letters 168 (2001) 141±144
www.elsevier.com/locate/canlet
Pro-apoptotic interactions between XK469 and the peripheral benzodiazepine receptor David Kessel*, Jerome P. Horwitz Departments of Pharmacology and Medicine, Wayne State University School of Medicine, Detroit MI 48201, USA Received 17 January 2001; received in revised form 21 March 2001; accepted 23 March 2001
Abstract XK469 (2-[4-(7-chloro-2-quinoxalinyloxy) phenoxy]propionic acid) is a new anti-tumor agent with substantial activity against several drug-resistant cell lines. Using murine leukemia L1210 cells in culture, we found the chiral R(1) form of XK469 to be substantially more cytotoxic than the S(2) form, while the herbicide analog `Assure w' was essentially inactive. The cytotoxic response to these agents was accompanied by apoptosis, and was found to be correlated with drug binding to the peripheral benzodiazepine receptor in cell culture, suggesting that receptor binding may be a factor in drug-induced cytotoxicity. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Peripheral benzodiazepine receptor; PK11195; XK469
1. Introduction The synthetic anti-tumor agent XK469 has a substantial anti-tumor activity against several solid tumor lines in culture and in vivo [1,2]. This drug was identi®ed during the screening of a large group of analogs of the herbicide Assure w, a product of the DuPont Chemical Co. A mechanism of action for XK469 was proposed in 1999 when it was determined that levels of XK469 in the mM range could inhibit topoisomerase IIb in a human ®broblast (GM637) cell line. While the chiral R form of XK469 was a more potent inhibitor than the S form, no studies on Assure w were carried out, so it was not clear whether the proposed mechanism could delineate between cytotoxic and inactive analogs. The present study was suggested by our ®nding that XK469 elicited an apoptotic response in the murine * Corresponding author. E-mail address: [email protected] (D. Kessel).
leukemia L1210 cell line. In previous studies [3,4], we had found that ligands for the peripheral benzodiazepine receptor could trigger such a apoptotic response. We therefore assessed the ability of the R(1) and S(2) forms of XK469 to compete with the isoquinoline PBR agonist PK11195 for binding. 2. Materials and methods 2.1. Chemicals The chiral forms of XK469 were provided by the National Cancer Institute. Solutions were prepared in DMSO and stored at 2108C. PK 11195 [2-chlorophenyl-N-methyl-N-(1methylpropyl)-3-isoquinolinecarboxamide] was purchased from Sigma Chemical Co., St Louis, MO. [N-methyl 3H]-PK 11195 (90 mC/mmol) was obtained from NEN Life Science Products, Boston, MA. This was diluted with carrier to a ®nal concentration of 1 mM.
0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00518-3
142
D. Kessel, J.P. Horwitz / Cancer Letters 168 (2001) 141±144
2.2. Cell cultures Murine leukemia L1210 cells were maintained in Fischer's Medium (Gibco, Grand Island NY) supplemented with 10% horse serum, 1 mM glutamine, 1 mM mercaptoethanol and gentamicin. Short-term incubations were carried out at a cell density of 7 mg/ml (wet weight) in a modi®ed growth medium, with HEPES (pH 7.4) replacing NaHCO3. Control and treated suspensions of L1210 cells were serially diluted, mixed with soft agar, plated, and cultured at 378C in a humidi®ed 5% CO2 chamber. Colonies were counted 5±7 days later. 2.3. Drug-induced apoptosis and cytotoxicity L1210 cells were incubated with speci®ed levels of XK469 (chiral forms) or Assure w at speci®ed levels for 4 h at 378C, then transferred to fresh medium for 16 h for assessing the apoptotic response. In other experiments, cells were treated with 500 nM staurosporin for 1 h at 378C, then incubated at the same temperature for 16 h. Apoptotic nuclei were labeled with HO33342 for examination by ¯uorescence microscopy as described in Ref. [5]. 2.4. Fluorescent probes Drug effects on the mitochondrial membrane potential (KCm) and on nuclear morphology were determined by labeling cells for 5 min at 378C with Mitotracker Orange (10 mg/ml) 1 HO33342 (5 mM). MTO ¯uorescence (580±620 nm) was detected upon excitation at 510±560 nm; HO342 ¯uoresence at 400±480 nm was detected upon excitation at 360±380 nm. All ¯uorescence studies were carried out using a Nikon E600 microscope ®tted with a digital CCD camera (Photometrics, Tucson, AZ). Images were processed with MetaMorph software (Universal Imaging Corp., West Chester, PA). 2.5. Binding studies Binding to the peripheral benzodiazepine receptor was determined by incubating cells for 30 min at 208C with 1 nM [ 13H]-PK11195 along with graded levels of XK469 or Assure w. The cells were then collected by centrifugation (800 £ g, 30 s) and washed once with cold isotonic NaCl. Radioactivity in cell pellets was
assessed by liquid scintillation counting. A plot of PK11195 binding vs. log [drug concentration] was used to determine the KD value for different agents. This represents the drug level needed to reduce PK11195 binding by 50%. 3. Results 3.1. Cytotoxicity studies L1210 cells were incubated with graded levels of XK469 (chiral forms) or Assure w as described above. Under these conditions, we found the R(1) form to be substantially more cytotoxic than S(2), with Assure w essentially non-toxic (Table 1). 3.2. Effects of XK469 and staurosporin on KC m and nuclear morphology Exposure of L1210 cells to a 7 mM concentration of the R (1) form of XK469 for 4 h led to a 50% loss of viability. This was accompanied by an apoptotic morphology that could be detected with the nuclear label HO 33342. The labeling pattern of a typical control (untreated) cell nucleus is shown in Fig. 1A. The apoptotic pattern obtained by treatment with staurosporin is shown in Fig. 1B. The nuclear labeling pattern of the apoptotic cells after exposure to XK469 was unusual with substantially more fragmentation detected (Fig. 1C). Additional studies were carried out to assess the loss of mitochondrial membrane potential (KCm) of apoptotic cells after treatment with XK469 for 4 h (Fig. 2). Cells with apoptotic nuclear morphology (Fig. 2A) uniformly showed a loss of KCm as detected Table 1 Growth inhibition Drug
LD50 value a
XK469 R(1) XK469 S(2) Assure w
7 ^ 2.6 83 ^ 6.4 . 500
a Murine leukemia L1210 cells were treated with graded levels of drugs for 4 h, then washed and incubated in growth medium for 4 days. The resulting cell populations were assessed using a Coulter counter, and the drug level (mM) needed to inhibit growth by 50% determined.
D. Kessel, J.P. Horwitz / Cancer Letters 168 (2001) 141±144
143
Fig. 1. HO33342 labeling patterns of L1210 nuclei. (A) Control; (B) apoptosis after treatment with 500 nM staurosporin; (C) apoptosis after exposure to 10 mM of the R(1) form of XK469.
by with the ¯uorescent probe MTO (Fig. 2B). Cells with a normal nuclear labeling pattern showed a typical mitochondrial staining pattern. 3.3. Receptor binding results A plot of log drug concentration vs. PK11195 binding is shown in Fig. 3. From these data, we calculated mean KD values for the R(1) form of XK469 0.35 mM, the S(2) form 0.12 mM and Assure w 1.1 mM. 4. Discussion In this study, we examined the ability of the R(1) and S(2) forms of XK469, along with the inactive analog Assure w, to serve as ligands for the peripheral benzodiazepine receptor in L1210 cells. These studies were prompted by the observation that exposure of L1210 cells to an LD90 concentration of XK469 led to an unusual apoptotic nuclear morphology and a loss of KCm. These data alone do not necessarily implicate the PBR as a factor in the apoptotic response.
Fig. 2. Labeling patterns of L1210 cells exposed to 10 mM XK469 (R) for 4 h. (A) HO342 labeling showing apoptotic nuclei; (B) MTO labeling showing loss of KCm.
Previous reports had indicated that the dark toxicity of porphyrin analogs led to an apoptotic response [3,4], and that porphyrins were considered to be `natural' ligands for the PBR [7]. Moreover, PK11195, another PBR ligand, was found to exert pro-apoptotic effects [8]. Competition studies with radioactive PK11195 revealed that the relative af®nity of the agents tested was correlated with cytotoxicity in cell culture (Table 1). These results are consistent with the hypothesis that af®nity for the PBR be a determinant of XK469 cytotoxicity. Adverse reactions to XK469 have not yet been identi®ed in normal host tissues, so we cannot make any conclusions regarding the role of PBR binding in the context of host toxicity. Like many other antitumor agents, XK469 may have multiple sites of action. Gao et al. [6] found evidence that the drug is an inhibitor of topoisomerase IIb. This result may explain the nuclear fragmentation
Fig. 3. Plot of % PK 11195 bound to cells vs. log concentration, comparing the R(1) and S(2) forms of XK469 with the analog Assure w.
144
D. Kessel, J.P. Horwitz / Cancer Letters 168 (2001) 141±144
associated with XK469 toxicity, but it is noteworthy that studies on the inactive analog Assure w were not carried out, and the XK469 levels used (0.5±1 mM) in the cell-free systems may be substantially greater than the intracellular concentration of this agent under LD50 conditions. Acknowledgements
[3] [4]
This work was supported in part by grant CA 82341 from the NIH. We thank Ann Marie Santiago and Brendan Leeson for excellent technical assistance.
[5]
References
[7]
[1] P.M. LoRusso, R. Parchment, L. Demchik, J. Knight, L. Polin, J. Dzubow, C. Behrens, B. Harrison, G. Trainor, T.H. Corbett, Preclinical antitumor activity of XK469 (NSC 656889) concentration dependent toxicity from 0.5±30 mg/mL, Invest. New Drugs 16 (1998) 287±296. [2] T.H. Corbett, P. LoRusso, L. Demchick, C. Simpson, S. Pugh,
[6]
[8]
K. White, J. Kushner, L. Polin, J. Meyer, J. Czarnecki, L. Heilbrun, J.P. Horwitz, J.L. Gross, C.H. Behrens, B.A. Harrison, R.J. McRipley, G. Trainor, Preclinical antitumor ef®cacy of analogs of XK469: sodium-(2-[4-(7-chloro-2-quinoxalinyloxy)phenoxy]propionate, Invest. New Drugs 16 (1998) 129± 139. D. Kessel, Y. Luo, Mitochondrial photodamage and PDTinduced apoptosis, J. Photochem. Photobiol. 42 (1998) 89±95. D. Kessel, Y. Luo, K. Woodburn, C.K. Chang, B. Henderson, Mechanism of phototoxicity catalyzed by two porphycenes, Proc. SPIE 2392 (1995) 122±128. D. Kessel, Y. Luo, Photodynamic therapy: a mitochondrial inducer of apoptosis, Cell Death Differ. 6 (1999) 28±35. H. Gao, K.C. Huang, E.F. Yamasaki, K.K. Chan, L. Chohan, R.M. Snapka, XK469, a selective topoisomerase IIb beta poison (Tox at 1±5 mM), Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 12168±12173. A. Verma, J.S. Nye, S.H. Snyder, Porphyrins are endogenous ligands for the mitochondrial (peripheraltype) benzodiazepine receptor, Proc. Natl. Acad. Sci. U.S.A. 84 (1987) 2256±2260. T. Hirsch, D. Decaudin, S.A. Susin, P. Marchetti, N. Larochette, M. Resche-Rigon, G. Kroemer, PK11195, a ligand of the mitochondrial benzodiazepine receptor, facilitates the induction of apoptosis and reverses Bcl-2-mediated cytoprotection, Exp. Cell Res. 241 (1998) 426±434.
www.elsevier.com/locate/canlet
Pro-apoptotic interactions between XK469 and the peripheral benzodiazepine receptor David Kessel*, Jerome P. Horwitz Departments of Pharmacology and Medicine, Wayne State University School of Medicine, Detroit MI 48201, USA Received 17 January 2001; received in revised form 21 March 2001; accepted 23 March 2001
Abstract XK469 (2-[4-(7-chloro-2-quinoxalinyloxy) phenoxy]propionic acid) is a new anti-tumor agent with substantial activity against several drug-resistant cell lines. Using murine leukemia L1210 cells in culture, we found the chiral R(1) form of XK469 to be substantially more cytotoxic than the S(2) form, while the herbicide analog `Assure w' was essentially inactive. The cytotoxic response to these agents was accompanied by apoptosis, and was found to be correlated with drug binding to the peripheral benzodiazepine receptor in cell culture, suggesting that receptor binding may be a factor in drug-induced cytotoxicity. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Peripheral benzodiazepine receptor; PK11195; XK469
1. Introduction The synthetic anti-tumor agent XK469 has a substantial anti-tumor activity against several solid tumor lines in culture and in vivo [1,2]. This drug was identi®ed during the screening of a large group of analogs of the herbicide Assure w, a product of the DuPont Chemical Co. A mechanism of action for XK469 was proposed in 1999 when it was determined that levels of XK469 in the mM range could inhibit topoisomerase IIb in a human ®broblast (GM637) cell line. While the chiral R form of XK469 was a more potent inhibitor than the S form, no studies on Assure w were carried out, so it was not clear whether the proposed mechanism could delineate between cytotoxic and inactive analogs. The present study was suggested by our ®nding that XK469 elicited an apoptotic response in the murine * Corresponding author. E-mail address: [email protected] (D. Kessel).
leukemia L1210 cell line. In previous studies [3,4], we had found that ligands for the peripheral benzodiazepine receptor could trigger such a apoptotic response. We therefore assessed the ability of the R(1) and S(2) forms of XK469 to compete with the isoquinoline PBR agonist PK11195 for binding. 2. Materials and methods 2.1. Chemicals The chiral forms of XK469 were provided by the National Cancer Institute. Solutions were prepared in DMSO and stored at 2108C. PK 11195 [2-chlorophenyl-N-methyl-N-(1methylpropyl)-3-isoquinolinecarboxamide] was purchased from Sigma Chemical Co., St Louis, MO. [N-methyl 3H]-PK 11195 (90 mC/mmol) was obtained from NEN Life Science Products, Boston, MA. This was diluted with carrier to a ®nal concentration of 1 mM.
0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00518-3
142
D. Kessel, J.P. Horwitz / Cancer Letters 168 (2001) 141±144
2.2. Cell cultures Murine leukemia L1210 cells were maintained in Fischer's Medium (Gibco, Grand Island NY) supplemented with 10% horse serum, 1 mM glutamine, 1 mM mercaptoethanol and gentamicin. Short-term incubations were carried out at a cell density of 7 mg/ml (wet weight) in a modi®ed growth medium, with HEPES (pH 7.4) replacing NaHCO3. Control and treated suspensions of L1210 cells were serially diluted, mixed with soft agar, plated, and cultured at 378C in a humidi®ed 5% CO2 chamber. Colonies were counted 5±7 days later. 2.3. Drug-induced apoptosis and cytotoxicity L1210 cells were incubated with speci®ed levels of XK469 (chiral forms) or Assure w at speci®ed levels for 4 h at 378C, then transferred to fresh medium for 16 h for assessing the apoptotic response. In other experiments, cells were treated with 500 nM staurosporin for 1 h at 378C, then incubated at the same temperature for 16 h. Apoptotic nuclei were labeled with HO33342 for examination by ¯uorescence microscopy as described in Ref. [5]. 2.4. Fluorescent probes Drug effects on the mitochondrial membrane potential (KCm) and on nuclear morphology were determined by labeling cells for 5 min at 378C with Mitotracker Orange (10 mg/ml) 1 HO33342 (5 mM). MTO ¯uorescence (580±620 nm) was detected upon excitation at 510±560 nm; HO342 ¯uoresence at 400±480 nm was detected upon excitation at 360±380 nm. All ¯uorescence studies were carried out using a Nikon E600 microscope ®tted with a digital CCD camera (Photometrics, Tucson, AZ). Images were processed with MetaMorph software (Universal Imaging Corp., West Chester, PA). 2.5. Binding studies Binding to the peripheral benzodiazepine receptor was determined by incubating cells for 30 min at 208C with 1 nM [ 13H]-PK11195 along with graded levels of XK469 or Assure w. The cells were then collected by centrifugation (800 £ g, 30 s) and washed once with cold isotonic NaCl. Radioactivity in cell pellets was
assessed by liquid scintillation counting. A plot of PK11195 binding vs. log [drug concentration] was used to determine the KD value for different agents. This represents the drug level needed to reduce PK11195 binding by 50%. 3. Results 3.1. Cytotoxicity studies L1210 cells were incubated with graded levels of XK469 (chiral forms) or Assure w as described above. Under these conditions, we found the R(1) form to be substantially more cytotoxic than S(2), with Assure w essentially non-toxic (Table 1). 3.2. Effects of XK469 and staurosporin on KC m and nuclear morphology Exposure of L1210 cells to a 7 mM concentration of the R (1) form of XK469 for 4 h led to a 50% loss of viability. This was accompanied by an apoptotic morphology that could be detected with the nuclear label HO 33342. The labeling pattern of a typical control (untreated) cell nucleus is shown in Fig. 1A. The apoptotic pattern obtained by treatment with staurosporin is shown in Fig. 1B. The nuclear labeling pattern of the apoptotic cells after exposure to XK469 was unusual with substantially more fragmentation detected (Fig. 1C). Additional studies were carried out to assess the loss of mitochondrial membrane potential (KCm) of apoptotic cells after treatment with XK469 for 4 h (Fig. 2). Cells with apoptotic nuclear morphology (Fig. 2A) uniformly showed a loss of KCm as detected Table 1 Growth inhibition Drug
LD50 value a
XK469 R(1) XK469 S(2) Assure w
7 ^ 2.6 83 ^ 6.4 . 500
a Murine leukemia L1210 cells were treated with graded levels of drugs for 4 h, then washed and incubated in growth medium for 4 days. The resulting cell populations were assessed using a Coulter counter, and the drug level (mM) needed to inhibit growth by 50% determined.
D. Kessel, J.P. Horwitz / Cancer Letters 168 (2001) 141±144
143
Fig. 1. HO33342 labeling patterns of L1210 nuclei. (A) Control; (B) apoptosis after treatment with 500 nM staurosporin; (C) apoptosis after exposure to 10 mM of the R(1) form of XK469.
by with the ¯uorescent probe MTO (Fig. 2B). Cells with a normal nuclear labeling pattern showed a typical mitochondrial staining pattern. 3.3. Receptor binding results A plot of log drug concentration vs. PK11195 binding is shown in Fig. 3. From these data, we calculated mean KD values for the R(1) form of XK469 0.35 mM, the S(2) form 0.12 mM and Assure w 1.1 mM. 4. Discussion In this study, we examined the ability of the R(1) and S(2) forms of XK469, along with the inactive analog Assure w, to serve as ligands for the peripheral benzodiazepine receptor in L1210 cells. These studies were prompted by the observation that exposure of L1210 cells to an LD90 concentration of XK469 led to an unusual apoptotic nuclear morphology and a loss of KCm. These data alone do not necessarily implicate the PBR as a factor in the apoptotic response.
Fig. 2. Labeling patterns of L1210 cells exposed to 10 mM XK469 (R) for 4 h. (A) HO342 labeling showing apoptotic nuclei; (B) MTO labeling showing loss of KCm.
Previous reports had indicated that the dark toxicity of porphyrin analogs led to an apoptotic response [3,4], and that porphyrins were considered to be `natural' ligands for the PBR [7]. Moreover, PK11195, another PBR ligand, was found to exert pro-apoptotic effects [8]. Competition studies with radioactive PK11195 revealed that the relative af®nity of the agents tested was correlated with cytotoxicity in cell culture (Table 1). These results are consistent with the hypothesis that af®nity for the PBR be a determinant of XK469 cytotoxicity. Adverse reactions to XK469 have not yet been identi®ed in normal host tissues, so we cannot make any conclusions regarding the role of PBR binding in the context of host toxicity. Like many other antitumor agents, XK469 may have multiple sites of action. Gao et al. [6] found evidence that the drug is an inhibitor of topoisomerase IIb. This result may explain the nuclear fragmentation
Fig. 3. Plot of % PK 11195 bound to cells vs. log concentration, comparing the R(1) and S(2) forms of XK469 with the analog Assure w.
144
D. Kessel, J.P. Horwitz / Cancer Letters 168 (2001) 141±144
associated with XK469 toxicity, but it is noteworthy that studies on the inactive analog Assure w were not carried out, and the XK469 levels used (0.5±1 mM) in the cell-free systems may be substantially greater than the intracellular concentration of this agent under LD50 conditions. Acknowledgements
[3] [4]
This work was supported in part by grant CA 82341 from the NIH. We thank Ann Marie Santiago and Brendan Leeson for excellent technical assistance.
[5]
References
[7]
[1] P.M. LoRusso, R. Parchment, L. Demchik, J. Knight, L. Polin, J. Dzubow, C. Behrens, B. Harrison, G. Trainor, T.H. Corbett, Preclinical antitumor activity of XK469 (NSC 656889) concentration dependent toxicity from 0.5±30 mg/mL, Invest. New Drugs 16 (1998) 287±296. [2] T.H. Corbett, P. LoRusso, L. Demchick, C. Simpson, S. Pugh,
[6]
[8]
K. White, J. Kushner, L. Polin, J. Meyer, J. Czarnecki, L. Heilbrun, J.P. Horwitz, J.L. Gross, C.H. Behrens, B.A. Harrison, R.J. McRipley, G. Trainor, Preclinical antitumor ef®cacy of analogs of XK469: sodium-(2-[4-(7-chloro-2-quinoxalinyloxy)phenoxy]propionate, Invest. New Drugs 16 (1998) 129± 139. D. Kessel, Y. Luo, Mitochondrial photodamage and PDTinduced apoptosis, J. Photochem. Photobiol. 42 (1998) 89±95. D. Kessel, Y. Luo, K. Woodburn, C.K. Chang, B. Henderson, Mechanism of phototoxicity catalyzed by two porphycenes, Proc. SPIE 2392 (1995) 122±128. D. Kessel, Y. Luo, Photodynamic therapy: a mitochondrial inducer of apoptosis, Cell Death Differ. 6 (1999) 28±35. H. Gao, K.C. Huang, E.F. Yamasaki, K.K. Chan, L. Chohan, R.M. Snapka, XK469, a selective topoisomerase IIb beta poison (Tox at 1±5 mM), Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 12168±12173. A. Verma, J.S. Nye, S.H. Snyder, Porphyrins are endogenous ligands for the mitochondrial (peripheraltype) benzodiazepine receptor, Proc. Natl. Acad. Sci. U.S.A. 84 (1987) 2256±2260. T. Hirsch, D. Decaudin, S.A. Susin, P. Marchetti, N. Larochette, M. Resche-Rigon, G. Kroemer, PK11195, a ligand of the mitochondrial benzodiazepine receptor, facilitates the induction of apoptosis and reverses Bcl-2-mediated cytoprotection, Exp. Cell Res. 241 (1998) 426±434.