DNA INTERACTION AND CYTOSTATIC ACTIVITY OF THE NEW LIVER ORGANOTROPIC COMPLEX OF CISPLATIN WITH GLYCOCHOLIC ACID: BAMET-R2 Jose J.G. MARIN 1 *, Rocio I.R. MACIAS 1 , Julio J. CRIADO 2 , Avelino BUENO 3 , Maria J. MONTE 1 and Maria A. SERRANO 4 1 Department of Physiology and Pharmacology, University of Salamanca, Salamanca, Spain 2 Department of Inorganic Chemistry, University of Salamanca, Salamanca, Spain 3 Department of Microbiology and Genetics, University of Salamanca, Salamanca, Spain 4 Department of Biochemistry and Molecular Biology, University of Salamanca, Salamanca, Spain The aim of this study was to investigate the ability of the new liver organotropic complex of cisplatin with glycocholate (GC), Bamet-R2, to interact with DN A, inhibit its replication and hence reduce tumor-cell proliferation. Changes in the electrophoretic mobility of the open and covalently closed circular forms of the pUC18 plasmid DNA from Escherichia coli, a shift in the denaturation temperature of double- stranded DN A, and ethidium-bromide displacement from DNA binding, were induced by Bamet-R2 and cisplatin, but not by GC. Neutral-red retention was used to measure the number of living cells in culture after long-term (72-hr) exposure to these compounds and to evaluate the effect on cell viability after short-term (6-hr) exposure. Bamet-R2 and cisplatin, but not GC, induced significant inhibition of cell growth. This effect ranged from mild to strong, depending upon the sensitivity of the different cell types as follows: cisplatin, rat hepatocytes in primary culture F rat hepatoma McA-RH7777 cells (rH) F human colon carcinoma LS 174T cells(hCC) F mouse hepatoma H epa 1–6 cells (mH ); Bamet- R2, rat hepatocytes F mH  hCC F rH . DN A synthesis was measured by radiolabeled-thymidine incorporation into DN A. Bamet-R2 and cisplatin, but not GC, significantly inhibited the rate of DNA synthesis by these cells. After short-term exposure to Bamet-R2 or GC, no acute cell toxicity was observed, except on hCC cells. By contrast, acute toxicity was induced by cisplatin for all cell types studied. T he in vivo anti-tumoral effect was investigated in 3 different strains of mice following s.c. implantation of tumor cells (mouse sar- coma S-180II cells in Swiss and B6 mice and hCC cells in nude mice). In all 3 models, tumor growth was inhibited by Bamet-R2 and cisplatin to a similar degree. H owever, signs of toxicity (increases in blood urea concentrations and de- creases in packed blood cell volume and in liver, kidney and body weight) and a reduction in survival rate were observed only during cisplatin administration. In sum, these results indicate that this bile-acid derivative can be considered as a cytostatic drug whose potential usefulness deserves further investigation. Int. J. Cancer 78:346–352, 1998.  1998 Wiley-Liss, Inc. Several strategies have been envisaged either to circumvent resistance to cytostatic drugs (Canon et al., 1990) or to improve their vectoriality towards tumor cells (Konno, 1992), which might reduce their toxicity towards extra-tumoral tissues. In view of the marked hepatobiliary organotropism of bile acids (Hofmann, 1994), these endogenous compounds or their analogs have been proposed as shuttles for drugs towards the liver (Ho, 1987; Betebenner et al., 1991; Stephan et al., 1992; Kramer et al., 1992; 1994). Owing to the amphipathic nature of bile acids (Carey, 1985), these compounds have also been used to modify the solubility properties of drugs (Maeda et al., 1990). The platinum(II)- containing bile acid derivatives synthesized by our group combine both aims, i.e., improvement in solubility (Criado et al., 1997a,b); and in hepatobiliary vectoriality (Marin et al., 1996b). These compounds have been designated as Bamet, ‘‘Ba’’standing for bile acid and ‘‘met’’standing for metal, since not only platinum but also other metals such as palladium and gold can be used to obtain different members of this family (Criado et al., 1997a,b). Prelimi- nary studies on one of these compounds, named Bamet-R2, afforded promising results as regards the cytostatic effect together with liver organotropism (Criado et al., 1997b). Platinum-derived drugs are believed to inhibit DNA synthesis in rapidly growing cells, such as cancer cells, by binding to DNA. The formation of these adducts alters DNA structure in a such way that replication either cannot proceed or results in non-viable daughter cells (Donaldson et al., 1994). The specificity of platinum(II)-atom binding to DNA and different possible binding sites have been reported (for review, see Sundquist and Lippard, 1990). The aim of the present work was therefore to further investigate the ability of this cisplatin/bile-acid complex to interact with DNA, inhibit DNA replication, and hence reduce tumor-cell proliferation. MATERIAL AND METHODS Chemicals Glycocholic acid (GC) sodium salt more than 95% pure by thin-layer chromatography, cisplatin, ethidium bromide (EthBr), neutral red (3-amino-7-dimethylamino-2-methyl-phenazine hydro- chloride), DNA from herring sperm, MES (2-[N-morpholino]eth- anesulfonic acid) and culture media were purchased from Sigma (St. Louis, MO). [ 14 C]-thymidine was obtained from New England Nuclear (Itisa, Madrid). Bamet-R2 [cis-diamminechlorocholylgly- cinateplatinum(II)] was synthesized and chemically characterized (see inset of Fig. 1) as reported (Criado et al., 1997b). DNA-drug interaction To investigate drug-induced changes in the electrophoretic mobility of a plasmid DNA (Sambrook et al., 1989) the drug under investigation was dissolved (325 μM) in 1 mM MES buffer, pH 6.0. This was mixed with a smaller volume (9:1; vol/vol) of TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8.0) containing pUC18 plasmid DNA purified from Escherichia coli (1 μg/μl). The amount of drug in the final solution was adjusted in order to achieve the desired drug/DNA per nucleotide ratio (Rf). After incubation in the dark for 24 hr, 20 μl-aliquots of this solution were used for electrophoresis at 75 V in 1% agarose gel prepared in 40 mM Tris-HCl, 1 mM EDTA, 20 mM acetic acid, pH 8.0. Ethidium- bromide solution (0.5 μg/ml) in a similar buffer was used to stain the gels for subsequent photography. Drug-induced changes in the DNA melting temperature were investigated as described by Church et al. (1993) using 5 mg/100 ml DNA from herring sperm in 5 mM Tris-HCl, pH 7.5, containing 1 mM NaCl. The solution was sonicated for 4 hr in an ice-cold bath. DNA concentrations were determined by UV absorption at 260 nm assuming a molar extinction coefficient () of 6600 M -1 cm -1 and Grant sponsor: CICYT; Grant number: SAF96-0146. *Correspondence to: Departamento de Fisiologia y Farmacologia, Cam- pus Miguel de Unamuno, ED-S09, 37007-Salamanca, Spain. Fax: (34)923- 294669. E-mail: jjgmarin@gugu.usal.es Received 2 March 1998; Revised 11 May 1998 Int. J. Cancer: 78, 346–352 (1998)  1998 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer