Potent induction of apoptosis in human hepatoma cell lines by targeted cytotoxic somatostatin analogue AN-238 Malika Lasfer 1 , Nathalie Vadrot 1 , Andrew V. Schally 2,3 , Attila Nagy 2,3 , Gabor Halmos 3,4 , Dominique Pessayre 1 , Ge´rard Feldmann 1 , Florence J. Reyl-Desmars 1, * 1 INSERM Unit 481, Faculte´ de Me´decine Xavier Bichat, IFR 02, 16 rue Henri Huchard, BP 416, 75870, Paris Cedex 18, France 2 Endocrine, Polypeptide and Cancer Institute Veterans Affairs Medical Center, Tulane University School of Medicine, New Orleans, LA 70112, USA 3 Section of Experimental Medicine, Department of Medicine, Tulane University School of Medicine, New Orleans, LA 70112, USA 4 Department of Biopharmacy, University of Debrecen, Medical and Health Science Center, H-4032 Debrecen, Hungary Background/Aims: The efficacy of a targeted cytotoxic hybrid somatostatin analogue AN-238 and of its superactive radical 2-pyrrolinodoxorubicin (AN-201) to induce apoptosis of HepG2 and Hep3B human hepatoma cell lines were studied. AN-238 was designed to selectively target tumor cells expressing somatostatin receptor subtypes (sst s ). Its effects on HepG2 or Hep3B cells displaying or lacking tumor suppressor p53, respectively, were compared. Normal rat isolated hepatocytes were also tested. Methods: sst s were characterized by binding assays and RT-PCR. Cytotoxicity was quantified by flow cytometry. DNA fragmentation was studied by gel electrophoresis, PARP cleavage by Western blot and ROS formation using fluorescent probes. Results: Specific binding of iodinated RC-160 to HepG2 and Hep3B cells, and its displacement by AN-238 was characterized. mRNA for hsst 2A was found in both cell lines. Flow cytometry showed a stronger effect of AN-238 than AN-201 to induce sub-G1 phase. DNA fragmentation, nuclear bodies, and PARP cleavage were observed. In addition, AN-238 increased formation of ROS more potently than AN-201. However, no inductions of DNA fragmentation by AN-201 or AN-238 were observed on rat hepatocytes. Conclusions: Our results indicate that, in liver cancer, the cytotoxic somatostatin analogue AN-238 is a powerful agent that can induce apoptosis, through sst s and independently of p53. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Hepatocellular carcinoma; Chemotherapeutic drugs; Tumor targeting; Hybrid compounds; Receptors; Cell death 1. Introduction Hepatocellular carcinoma (HCC) is one of the common- est malignancies [1]. The therapy of choice with the best chance of cure is a surgical removal of the tumor. However, more than 70% of patients are diagnosed with non-resectable disease for which there is no effective therapy available at present [2,3]. Among the various treatment modalities, chemotherapeutic agents such as doxorubicin (DOX) have been investigated alone and in combination with other cytotoxic compounds in patients with HCC [3,4]. Unfortunately, they cause severe toxicity, especially in cirrhotic patients, and their efficacy is low, due to the multi-drug resistant nature of HCC [2]. Other treatment options also have very limited efficacies. Conse- quently, new modalities must be developed. Recent studies indicate that HCC patients may benefit from treatment with somatostatin analogues, such as octreotide and lanreotide [5–7]. They inhibit the proliferation of cancer cells in vitro through binding to hsst 2 >hsst 3 >hsst 5 [8,9]. Because binding sites for iodinated Tyr 3 -octreotide were found on 24 of 59 HCC biopsy Journal of Hepatology 42 (2005) 230–237 www.elsevier.com/locate/jhep 0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.10.014 Received 7 July 2004; received in revised form 1 October 2004; accepted 26 October 2004; available online 25 November 2004 * Corresponding author. Tel.: C33 1 44 85 61 98; fax: C33 1 44 85 92 79. E-mail address: reyl@bichat.inserm.fr (F.J. Reyl-Desmars).