Molecular and cellular pharmacologyImproved in vitro antitumor potential of (O,O′-Diisobutyl-ethylenediamine-N,N′-di-3-propionate)tetrachloridoplatinum(IV) complex under normoxic and hypoxic conditions
Graphical abstract
Introduction
One of the major causes of death in humans is cancer. Cisplatin, cis-[PtCl2(NH3)2], (Fig. 1A) is one of the most successful anticancer compounds in treatment of various types of tumors (Harrap, 1985, Kalinowska-Lis et al., 2008, Kidani et al., 1978, Knox et al., 1986, Lippert, 1999, Siddik, 2003, Rosenberg et al., 1965, Wong and Giandomenico, 1999). Since the discovery of its activity, unfortunately accompanied by serious side effects (e.g. nephrotoxicity, emetogenicity and neurotoxicity) synthesis of new platinum-based compounds that would be less toxic to healthy tissue gained a progressively increasing interest (Allardyce et al., 2005, Dyson and Sava, 2006, Garbutcheon-Singh et al., 2011, Gómez-Ruiz et al., 2012, Orvig and Abrams, 1999, Rau and van Eldik, 1996). About 20 million cancer cases are expected to occur in the next two decades, which renders the quest for new and improved antineoplastic agents.
This is an urgent issue in the field of Biomedicine and Human Health. Besides cisplatin, only carboplatin and oxaliplatin are in worldwide clinical use (Kelland, 2007). Particularly in the treatment of cancer, inorganic compounds have had an enormous impact, their activity relying mostly on specific interactions with DNA, leading to damage and ultimately to cell death (Dyson and Sava, 2006, Farrell, 1999, Farrell, 2004, Fricker, 2007, Gielen, 1988, Hannon, 2007, Van Rijt and Sadler, 2009).
There is a growing interest in platinum(IV) compounds as they have increased inertness relative to platinum(II) complexes. This property allows oral administration and reduced toxicity (Choy, 2006, Galanski et al., 2003, Hall and Hambley, 2002, Kelland, 2007, Zhang and Lippard, 2003). Satraplatin, as a platinum(IV) complex that has overcome Phase III clinical trials, showed great efficiency against several platinum-resistant human cancer cell lines (Choy et al., 2008, Fokkema et al., 2002, Samimi and Howell, 2006), including lung, ovary, cervix, and prostate (pivotal SPARC trial, Satraplatin and Prednisone Against Refractory Cancer) (Sonpavde and Sternberg, 2009, Sternberg et al., 2009). Proper choice of ligands is crucial for anticancer activity of metallodrugs, and it should be following structure–activity relationships. These organic molecules can modify lipophilicity or stabilize specific oxidation states of metal ions, as well as control reactivity (Arnesano and Natile, 2009).
Platinum(IV) complexes with R2edda-type ligands (dialkyl esters of ethylenediamine-N,N′-diacetic acid and its derivatives), are considered to be variants of satraplatin with κ2N,N′ mode of coordination (Kaluđerović et al., 2008b). Such compounds, obtained by structural variations of the aminocarboxylato arms and alkyl groups of the ester moiety (normal, branched chains, rings), yielded a large group of R2edda-type platinum complexes (Kaluđerović et al., 2008a, Kaluđerović et al., 2009, Krajčinović et al., 2008, Lazić et al., 2010, Vujić et al., 2011, Zmejkovski et al., 2009). The most efficient complexes were found to be tetrachloridoplatinum(IV) complexes with nBu and nPe esters of ethylenediamine-N,N′-di-3-propionic acid, [PtCl4(nBu2eddp)] and [PtCl4(nPe2eddp)] (Fig. 1B) against human adenocarcinoma HeLa cells (approx. five times less active than cisplatin) and human myelogenous leukemia K562 cells (comparable with cisplatin) acting through apoptotic cell death (Kaluđerović et al., 2005a). Noteworthy in vitro activity caused by these two platinum(IV) complexes was demonstrated on L929 fibrosarcoma and U251 astrocytoma tumor cells (Kaluđerović et al., 2005b, Mijatović et al., 2005). The compounds are found to induce considerably faster tumor cell death process than cisplatin (Kaluđerović et al., 2005b).
Recently, [PtCl4(iBu2eddp)] (Fig. 1C) was tested, among related compounds, against various cell lines: CT26CL25, HTC116, SW620, PC3, LNCaP, U251, A375, B16 and two normal primary cells (fibroblasts and keratinocytes) (Kaluđerović et al., 2012). On the representative HCT116 cell line, human colon cancer, mechanism of action was studied and it was found that it is less toxic to normal primary cells but induced caspase dependent apoptosis of HCT116 followed with autophagic cell death. Furthermore, reactive oxygen species and reactive nitrogen species are not the major mediators of drugs toxicity (Kaluđerović et al., 2012). In this work we report that [PtCl4(iBu2eddp)] complex showed qualitatively improved in vitro antitumor potential in comparison to cisplatin under normoxic and hypoxic conditions.
Section snippets
Synthesis of [PtCl4(iBu2eddp)]
The platinum(IV) complex, [PtCl4(iBu2eddp)], was synthesized as described in literature (Kaluđerović et al., 2012) and structure was confirmed with 1H and 13C NMR spectroscopy.
Reagents
Fetal calf serum (FCS), RPMI-1640, phosphate-buffered saline (PBS), and propidium iodide (PI) were obtained from Sigma (St. Louis, MO). Annexin V-FITC (AnnV) was from Biotium (Hayward, CA). Apostat was purchased from R&D (R&D Systems, Minneapolis, MN USA). Hypoxic bags (Microbiology Anaerocult A mini) were bought from
[PtCl4(iBu2eddp)] decreases cell viability more potently than cisplatin
L929 cells were exposed to a wide range of doses of cisplatin or [PtCl4(iBu2eddp)]. After 24 h of cultivation, both compounds remarkably decreased the number of viable cells (Fig. 2A). IC50 results revealed significant difference in effective concentration of tested compounds (IC50=27 µM, cisplatin; 16 µM, [PtCl4(iBu2eddp)]). Time dependent evaluation of the cell viability of cultures exposed to IC50 doses of platinum(IV) and platinum(II) complexes revealed difference already visible after only 2 h
Discussion
The in vitro antitumor activity of [PtCl4(iBu2eddp)] was investigated against mouse fibrosarcoma L929 cell line. The results showed dose-dependent action of tested platinum complex and higher efficiency in comparison to cisplatin against investigated cells (Fig. 2A). Concordantly with our previous data (Kaluđerović et al., 2012), it is obvious that [PtCl4(iBu2eddp)] complex possesses more potent capacity to down-regulate tumor cell growth than cisplatin. These two structurally different
Conclusion
In the current study, detailed analysis of [PtCl4(iBu2eddp)] antitumor action against fibrosarcoma L929 cell line was conducted and the following conclusions can be drawn:
- 1.
Platinum(IV) compound revealed improved anticancer profile in comparison to cisplatin.
- 2.
[PtCl4(iBu2eddp)] induced necrotic cell death accompanied with strong apoptotic process.
- 3.
This dual potential makes it selective toward cancer cells.
- 4.
[PtCl4(iBu2eddp)] induces less DNA damage and its toxicity is more related to ATP depletion,
Acknowledgments
The authors would like to acknowledge financial support from the Ministry of Science and Technological Development of the Republic of Serbia (Project nos. 173013, 173020 and 172035), SMWK (Project no. 33707045), SAB (ESF, Project no. 100147954) and the Free State of Saxony (Project no. 100099597) and Dr. Djordje Miljković for the help in flow cytometry analysis.
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