[CANCER RESEARCH 60, 4194 – 4199, August 1, 2000] Regulation of Targeted Chemotherapy with Cytotoxic Lutenizing Hormone- releasing Hormone Analogue by Epidermal Growth Factor 1 Linda J. Krebs, Xiaopeng Wang, Haridas E. Pudavar, Earl J. Bergey, Andrew V. Schally, Attila Nagy, Paras N. Prasad, and Charles Liebow 2 Department of Chemistry, Institute for Lasers, Photonics, and Biophotonics, State University of New York, Buffalo, New York 14260 [L. J. K., X. W., H. E. P., E. J. B., P. N. P., C. L.]; Departments of Physiology and Biophysics [L. J. K., C. L.] and Oral Biology [L. J. K.], State University of New York, Buffalo, New York 14214; and Veterans Affairs Medical Center and Tulane University School of Medicine, New Orleans, Louisiana 70112 [A. V. S., A. N.] ABSTRACT Targeting chemotherapy selectively to cancers can reduce the toxic side effects. AN-152, a conjugate of doxorubicin and [D-Lys 6 ]-luteiniz- ing hormone-releasing hormone (LH-RH), is more potent against LH-RH receptor-bearing cancers and produces less peripheral toxicity than doxorubicin. Many cancers, e.g., 50% of breast cancers, but few normal tissues express these receptors, providing a selective target for this cytotoxic conjugate. In this study, the effectiveness of AN-152 was heightened by receptor up-regulation. The cytotoxic effect of AN-152 can be regulated by the number of active LH-RH receptors on cancer cells. LH-RH receptor-positive (MCF-7) and -negative (UCI-107) can- cer cells were treated with epidermal growth factor (EGF) or the somatostatin analogue, RC-160. EGF and RC-160 have been shown previously to regulate LH-RH receptors through phosphorylation. The effect of receptor regulation, by hormone exposure, on the cytotoxicity of AN-152 and doxorubicin and on the cellular uptake of AN-152, [D-Lys 6 ]LH-RH, or doxorubicin was assessed by the 3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and by two-pho- ton laser scanning microscopy. The results demonstrated that the cellular entry of the conjugate was: (a) specific for cancers with LH-RH receptors; (b) up-regulated by EGF; (c) down-regulated by RC-160; and (d) the cytotoxicity of the AN-152 paralleled the efficiency of entry. This study illustrates the potential use of receptor regulation for increasing the efficacy of chemotherapeutic approaches that are directed to cell surface receptors. INTRODUCTION Chemotherapy is commonly used in the treatment of many cancers, but it has some distinct limitations and disadvantages (1). A major limitation is that it is not always efficacious because of severe restric- tions on the drug dosages that can be used (1). The dose of drug that is required to eradicate some cancers may not be compatible with patient survival. The disadvantage of chemotherapy is mainly related to the toxicity of the drug. The toxicity of the drug makes the patient susceptible to potentially fatal infections, cardiac toxicity, and other side effects. Reducing these side effects by targeting cytotoxic agents more selectively to cancer cells can improve the patients’ chances of a cure and make the quality of life more tolerable to the patient (1). The targeting of cytotoxic agents linked to hydrophilic peptide hormone analogues could help overcome some of the shortcomings of chemotherapy. AN-152 was developed by coupling the frequently used antineoplastic agent, doxorubicin, to an analogue of the peptide hormone LH-RH, 3 [D-Lys 6 ]LH-RH (2). This analogue was chosen to serve as a carrier peptide for targeting because many tumors express receptors for LH-RH (3–5). In addition to gonadotrope cells in the anterior pituitary, the expression of LH-RH receptors has been dem- onstrated in various human cancers, including breast (6), ovarian (7–9), endometrial (3, 8, 9), prostatic (4, 10, 11), and pancreatic (12, 13). AN-152 is significantly less toxic and more potent against can- cers with LH-RH receptors than doxorubicin (2, 14). The decreased toxicity is presumably caused by a slower entry into normal cells than doxorubicin, resulting in a different tissue distribution of the more hydrophilic peptide conjugate from that of the more lipophilic doxo- rubicin. The enhanced efficacy is likely attributable to the high- affinity binding of AN-152 to receptors for LH-RH (15) on these cancers. Thus, treatment of cancers expressing LH-RH receptors with AN-152 would be more selective and would exert a lower toxicity than conventional therapies. It has been proposed that the expression of LH-RH receptors in cancers is mediated by the loss of balance in the tyrosine kinase growth control pathway seen in many of these cancers (16, 17). In the hamster cheek pouch carcinoma model of oral cancer, LH-RH recep- tors appear in a predictable manner during carcinogenesis (16). This change takes place much more rapidly by enhancing selective tyrosine kinase activity through events mediated by EGF stimulation (16). Previous studies using cell membrane preparations have shown that LH-RH receptors can be activated by phosphorylation and inactivated by dephosphorylation (17). LH-RH receptor activation can be dramat- ically increased by EGF-stimulated tyrosine kinase activity (17). The increase in the tyrosine kinase signal in cancer cells results in activa- tion of latent receptors by phosphorylation and increases the number of functional receptors (17). An analogue of somatostatin, RC-160, which activates tyrosine phosphatase activity, decreases the number of functional receptors through dephosphorylation (17). The use of cell membrane extracts in these LH-RH binding experiments eliminated receptor synthesis or degradation as possible explanations for the modulation of receptor binding in response to EGF and RC-160 (17). Lee et al. (18) reported that EGF specifically phosphorylates a tyro- sine residue on a M r 60,000 protein, which corresponds to the LH-RH receptor (19), and that RC-160 dephosphorylates a tyrosine residue on that same protein (18). These findings indicate that the modulation of LH-RH receptor binding in cell membrane preparations in response to EGF and RC-160 are attributable to tyrosine kinase and tyrosine phosphatase stimulation, respectively. In this study, we show that acute treatment of cancer cells that express LH-RH receptors with EGF or RC-160 can sensitize or desensitize cells to AN-152 chemotherapy. To evaluate AN-152 ac- tion on cancer cells and modulation of its uptake by EGF and RC-160, the cytotoxic agent was conjugated to a two-photon fluorophore, C625, used to label AN-152 (AN-152:C625), [D-Lys 6 ]LH-RH ([D- Received 12/13/99; accepted 5/24/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Margaret Duffey-Cameron Troupe Foundation (Buffalo General Hospital), by the Dentist Scientist Award Grant 5K16DE00158, and by the Directorate of Chemistry and Life Sciences of the Air Force of Scientific Research Contract F496209710454. 2 To whom requests for reprints should be addressed, at Institute for Lasers, Photonics, and Biophotonics, Department of Chemistry, NSM Complex, Room 811, State University of New York, Buffalo, New York 14260-3000. E-mail: liebow@acsu.buffalo.edu. 3 The abbreviations used are: LH-RH, luteinizing hormone-releasing hormone; AN- 152, conjugate of [D-Lys 6 ]LH-RH and doxorubicin; EGF, epidermal growth factor; TPLSM, two-photon laser scanning microscopy; C625, 4-(N,N-diphenylamino)-4'-(6-O- hemiglutarate)hexylsulfinyl stilbene. 4194 Research. on September 17, 2015. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from