Systemic administration of a peptide that impairs the Protein Kinase (CK2) phosphorylation reduces solid tumor growth in mice Yasser Perera 1 , Hern an G. Farina 2 , Ignacio Hern andez 3 , Osmany Mendoza 1 , Joem M. Serrano 1 , Osvaldo Reyes 1 , Daniel E. G omez 2 , Roberto E. G omez 4 , Boris E. Acevedo 1 , Daniel F. Alonso 2 and Silvio E. Perea 1 * 1 Laboratory of Molecular Oncology, Division of Pharmaceuticals, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba 2 Laboratory of Molecular Oncology, Quilmes National University, Buenos Aires, Argentina 3 Development Department, Isotopes Center, Havana, Cuba 4 ELEA Laboratories, Buenos Aires, Argentina The antitumor efficacy of the CK2 inhibitors so far described has not been extensively evaluated in cancer animal models. We have previously demonstrated that a proapoptotic cyclic peptide termed P15 delivered into the cells by the Tat Cell Penetrating Peptide was able to abrogate the CK2-mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice. Here we explored the antitumor effect by systemic administration of P15-Tat in a consecutive 5-day schedule through either intraperitoneal or intravenous route. Importantly, signifi- cant delay of tumor growth was observed at 2 mg/kg (p < 0.05), 10 mg/kg (p < 0.01) or 40 mg/kg (p < 0.001) after P15-Tat adminis- tration both in syngeneic murine tumors and human tumors xeno- grafted in nude mice. In line with this, the systemic administration of P15-Tat induced apoptosis in the tumor as evidenced by in situ DNA fragmentation. Furthermore, we evidenced that 99m Tc-la- beled P15-Tat peptide was certainly accumulated on the tumors after administration by both routes. This report becomes the first describing the antitumor effect induced by systemic administra- tion of a peptide that targets the acidic phosphorylation domain for CK2 substrates. Also, our data reinforces the perspectives of P15-Tat for the cancer targeted therapy. ' 2007 Wiley-Liss, Inc. Key words: casein Kinase 2; cancer targeted therapy; tumor xenograft; cell penetrating peptide Exploitation of kinases as cancer therapeutic targets is continu- ously growing and its clinical validation becomes a reality as therapies with some specific inhibitors have shown clinical benefit in cancer patients. 1,2 Protein Kinase CK2 is a very conserved ser- ine/threonine kinase involved in the phosphorylation of a plethora of substrates, which display a very conserved acidic phosphoryla- tion domain. 3 CK2 is essential for different cell functions like gene expression, 4 cell growth, 5 cell survival, 6 chromatin remodel- ing 7 and protection of cells against apoptosis. 8 Further to the find- ings above mentioned the potential of CK2 as a particularly suitable target for cancer treatment has been experimentally validated by different groups. For example, studies in transgenic models of can- cer have demonstrated that CK2 overexpression displays a great on- cogenic potential and tumorigenicity. 9,10 Likewise, CK2 signal is uniformly dysregulated 3- to 7-fold in different cancer types 11 and also it has been associated with aggressive tumor behavior in human squamous cell carcinoma of head and neck cancer. 12 Other important hallmark that meets CK2 as an ideal target for cancer therapy is the cell death observed after its targeting by dif- ferent agents. For instance, condensed polyphenolic compounds, tetrabromobenzimidazole/triazole derivatives and indoloquinazo- lines have been shown to selectively inhibit the CK2 enzyme and exhibit a remarkable proapoptotic efficacy on a variety of tumor cell lines. 13 Similarly, CK2 down-regulation and antitumor effect have been observed using antisense CK2 a oligodeoxynucleotide in PC3-LN4 xenograft tumors in nude mice. 14 Thus, the develop- ment and exploitation of CK2 inhibitors could provide new hopes for cancer therapy. The anti-CK2 approaches so far described have been focused to target the enzyme itself either by biochemical or genetic proce- dures; however, we had previously described a peptide (P15-Tat) targeting the acidic phosphorylation domain for the CK2 sub- strates. 15 P15-Tat induced apoptosis on tumor cells and tumor regression when injected directly into solid tumors in mice. Although the direct administration into solid tumors has been used to evaluate anticancer drugs in animal models, 16,17 this route is not the only feasible choice for applying these kind of therapeu- tics. Data presented in this work reveal the feasibility of the sys- temic route as an alternative for P15-Tat administration in cancer animal models. Furthermore, our data reinforce the potential of a peptide that targets the acidic phosphorylation domain for CK2 substrates as a suitable compound for targeted cancer therapy. Material and methods Peptide synthesis P15-Tat was synthesized on solid phase and purified by reverse- phase high-performance liquid chromatography to >98% purity on acetonitrile/H 2 O-trifluoroacetic acid gradient and confirmed by ion-spray mass spectrometry (Micromass, Manchester, United Kingdom). In vivo therapy of solid tumors in mice C57/BL6 mice were subcutaneously injected with 1 3 10 5 TC-1 murine lung epithelial tumor cells per 300 lL of PBS on the right flank. After 10 days, before tumor debut, animals (n 5 8) received intraperitoneal daily injections for 5 consecutive days with 2.5, 10 or 40 mg/kg of P15-Tat or vehicle PBS in the control group. For human tumor xenografts, pathogen-free female athymic nu/ nu (nude) mice (Harlan, Germany/CNEA, Buenos Aires) were used at 6–8 weeks of age. In one of the therapeutic regimens, nude mice were subcutaneously injected with 5 3 10 6 NCI-H125 human non small cell lung cancer (NSCLC) cells per 300 lL of PBS on the right flank. Tumor-bearing animals (n 5 7) with 30 mm 3 received intraperitoneal injections for 5 consecutive days with 2 or 10 mg/kg of P15-Tat or vehicle PBS. Additionally, nude mice were subcutaneously injected with 1 3 10 7 HeLa human cer- vical adenocarcinoma cells per 400 lL of serum-free D-MEM on the right flank. After 4 days, before tumor debut, the P15-Tat pep- tide was administered daily at a dose of 20 mg/kg via the tail vein for 5 consecutive days (n 5 4). Animals were maintained in pathogen-free conditions and pro- cedures were performed in accordance with recommendations for Grant sponsors: Heber Biotec SA, Biorec. *Correspondence to: Laboratory of Molecular Oncology, Division of Pharmaceuticals, Center for Genetic Engineering and Biotechnology (CIGB), Havana CP10600, PO BOX 6162, Cuba. Fax: 53-7-271-8070. E-mail: silvio.perea@cigb.edu.cu Received 29 December 2006; Accepted after revision 21 June 2007 DOI 10.1002/ijc.23013 Published online 10 September 2007 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 122, 57–62 (2008) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer