Cytotoxicity of half sandwich ruthenium(II) complexes with strong hydrogen bond acceptor ligands and their mechanism of action Sangeeta Das a , Sarika Sinha b , Ramona Britto b , Kumaravel Somasundaram b, * , Ashoka G. Samuelson a, * a Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012, India b Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India article info Article history: Received 31 July 2009 Received in revised form 17 September 2009 Accepted 21 September 2009 Available online 8 October 2009 Keywords: Ruthenium(II) complexes DNA binding Cancer Cytotoxicity Microarrays Cell cycle analysis abstract Neutral and cationic organometallic ruthenium(II) piano stool complexes of the type [(g 6 -cymene)R- uCl(X)(Y)] (complexes R1–R8) has been synthesized and characterized. In cationic complexes, X, Y is either a g 2 phosphorus ligand such as 1,1-bis(diphenylphosphino)methane (DPPM) and 1,2-bis(diphen- ylphosphino)ethane (DPPE) or partially oxidized ligands such as 1,2-bis(diphenylphosphino)methane monooxide (DPPMO) and 1,2-bis(diphenylphosphino)ethane monooxide (DPPEO) which are strong hydrogen bond acceptors. In neutral complexes, X is chloride and Y is a monodentate phosphorous donor. Complexes with DPPM and DPPMO ligands ([(g 6 -cymene)Ru(g 2 -DPPM)Cl]PF 6 (R2), [(g 6 -cymene)Ru(g 2 - DPPMO)Cl]PF 6 (R3), [(g 6 -cymene)Ru(g 1 -DPPM)Cl 2 ](R5) and [(g 6 -cymene)Ru(g 1 -DPPMO)Cl 2 ](R6) show good cytotoxicity. Growth inhibition study of several human cancer cell lines by these complexes has been carried out. Mechanistic studies for R5 and R6 show that inhibition of cancer cell growth involves both cell cycle arrest and apoptosis induction. Using an apoptosis PCR array, we identified the sets of anti- apoptotic genes that were down regulated and pro-apoptotic genes that were up regulated. These com- plexes were also found to be potent metastasis inhibitors as they prevented cell invasion through matri- gel. The complexes were shown to bind DNA in a non intercalative fashion and cause unwinding of plasmid DNA in cell-free medium by competitive ethidium bromide binding, viscosity measurements, thermal denaturation and gel mobility shift assays. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Cisplatin is arguably the most widely used metallodrug for solid tumors, but its use has been limited due to the development of resistance in cancer cells and high toxicity to normal cells. There has been an accelerated search to identify new metal complexes with high cytotoxic property specific to cancer cells. Among the various metal complexes examined for cytotoxicity, organometal- lic ruthenium complexes have emerged as one of the most promis- ing class of compounds as they allow a great deal of molecular tinkering and the half sandwich complexes of Ru(II) provide an ex- tremely good scaffold for building new molecules. Research groups of Sadler, Dyson, Keppler and Reedijk have synthesized a remark- ably large number of Ru(II)/Ru(III) organometallic complexes that are being tested for anticancer activity [1–10]. Ruthenium com- plexes are quite cytotoxic but do not affect normal cells signifi- cantly [11,12]. The mechanistic studies carried out by these groups have iden- tified advantages in using ruthenium based metal complexes and their mode of action. The efficiency of ruthenium complexes lies in its ability to bind transferrin which is over expressed in the tu- mor cell thereby targeting cancer cells specifically [13,14]. The ability of cells to ‘‘expel” excess ruthenium which is similar to iron is suspected to be another reason for the lower toxicity of ruthe- nium based complexes compared to those based on platinum. It is now known that ruthenium can bind to DNA just as well as platinum and this might be a possible mode by which it induces cytotoxicity [15–18]. Ruthenium-chloro complexes tend to under- go hydrolysis in aqueous media leading to the generation of cat- ionic Ru–OH 2 complexes capable of reacting with DNA with greater ease than the corresponding chloro complexes [19–21]. The hydrolyzed complexes interact with the N7 of guanine in DNA duplexes leading to disruption of the structure of genetic material [21,22]. Sadler and coworkers have demonstrated that choice of aromatic ring systems in half sandwich complexes can attenuate the binding efficiency of these ruthenium complexes to DNA through p stacking [23]. Tetrahydroanthracene has been shown to be the best aromatic ligand for binding DNA [24]. Fur- thermore, they have shown that hydrogen bonding with the ethy- 0162-0134/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2009.09.017 * Corresponding authors. Tel.: +91 80 22932973; fax: +91 80 23602697 (K. Somasundaram), tel.: +91 80 22932663; fax: +91 80 22933355 (A.G. Samuelson). E-mail addresses: skumar@mcbl.iisc.ernet.in (K. Somasundaram), ashoka@ip- c.iisc.ernet.in (A.G. Samuelson). Journal of Inorganic Biochemistry 104 (2010) 93–104 Contents lists available at ScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio