Synthesis and In-vitro Evaluation of Anticancer Rh(III) Complexes Jun Liang, Aviva Levina, Peter A. Lay School of Chemistry, The University of Sydney, NSW 2006, Australia Email:
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INTRODUCTION The platinum complexes achieved spectacular success in cancer treatment, although their further application are hampered by severe dose-limiting side effects [1]. Such challenges lead to the development of metal-based chemotherapeutics with alternative metals, and a number of ruthenium complexes displayed superior pharmacology in early clinical trials [2]. These findings suggested that other platinum group metals might also possess anticancer properties, and a number of Rh(III) coordination and organometallic complexes have been reported to exhibit promising cytotoxicity and anti-metastatic effects [3]. The present study features the synthesis and in-vitro evaluation of a series of Rh(III) complexes with biologically relevant ligands, imidazole (Im) and indazole (Id). The antiproliferative activity of these complexes have been evaluated with MTT assay, and their intracellular accumulation profile were studied with atomic absorption spectroscopy (AAS). Their capability to inhibit cancer cell migration were studied with live cell imaging assay based on IncucyteTM system. Synchrotron X-ray absorption spectroscopy (XAS) was employed to provide further insight on the antimetastatic effects of the most promising drug candidates.
METHODS Synthesis of Rh complexes
Antiproliferative Assay (MTT) and Intracellular Metal Accumulation Study
IncucyteTM Live Cell Imaging Analysis
Synchrotron XAS Speciation •
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The Cys-Cys disulfide bond in native collagen I was reduced to free thiols with immobilized TCEP resin (TECP = Tris[2-carboxyethyl] phosphine hydrochloride); Rh complex 1 reacted with both native and reduce collagen I gel for 4h at 37 oC; Freeze dried
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The Rh complexes 1, 2, 3 and 4 were synthesised via literature procedures [4];
RESULTS AND DISCUSSIONS IncucyteTM Live Cell Imaging Analysis
Antiproliferative Assay (MTT) and Intracellular Metal Accumulation Study
DMSO 0.1%
NAMI-A 12.5 µM
NAMI-A 100 µM
1 12.5 µM
1 100 µM
0h
The IC50 values (half maximal inhibitory concentration) of Rh complexes and cisplatin determined with MTT assay Compounds Cancer Cell Line A2780 A2780CisR A549 1 42.07 ± 16.62 > 200 > 200 2 4.62 ± 0.32 8.04 ± 0.49 14.30 ± 9.98 3 0.095 ± 0.002 0.24 ± 0.01 5.33 ± 5.40 4 N/A N/A > 200 Cisplatin 0.93 ± 0.04 5.40 ± 0.47 2.23 ± 0.56
• Complexes 2 and 3 displayed pronounced cytotoxicity against all tested cell lines, 1 and 4 were only mildly cytotoxic; • Both 2 and 3 were less resisted by A2780CisR cell line than cisplatin, 3 exhibited sub-micromolar IC50 value on both cisplatin sensitive and resistant cell lines; • The cytotoxicity effect directly proportional to intracellular Rh concentration;
48 h
• Both NAMI-A and Rh complex 1 slowed down A549 cell migration into wound area; • Better suppression of A549 cell migration by Rh complex 1 at the lowest dose (12.5 µM) than those of NAMI-A at all the other doses tested. The highest dose (100 µM) of 1 caused cell rounding and regarded to be toxic; • The anticancer activity of Rh complex 1 possibly consisted of two aspects: the anti-metastatic property at low concentration and cytotoxicity at high concentration;
CONCLUSIONS
Synchrotron XAS Speciation • Shift in edge energy and increased average bond length for the parent pro-drug and its transformed product obtained from X-ray absorption spectra of Rh complex 1 in reduced collagen I gel indicated the presence of rhodiumsulfur bonds (Rh-S) [6]; • Formation of Rh-S bonds might be due to the ligand exchange between complex 1 and the free thiols in reduced collagen I gel; • Free thiols are abundant within the periphery of tumours due to elevated matrix metalloproteinase activity [7]; • The antimetastatic effect of Rh complex 1 is most likely due to its interactions with free thiols of the collagen network in tumour periphery, which renders the network less permeable to metastatic cancer cells [8]; References [1] Wheate, N. J. et, al, The status of platinum anticancer drugs in the clinic and in clinical trials. Dalton transactions 2010, 39 (35), 8113-27. [2] Bergamo, A. et. al., Approaching tumour therapy beyond platinum drugs: Status of the art and perspectives of ruthenium drug candidates. Journal of inorganic biochemistry 2012, 106 (1), 90-99. [3] Geldmacher, Y. et. al., Rhodium(III) and iridium(III) complexes as anticancer agents. Inorganica Chimica Acta 2012, 393, 84-102. [4] Mestroni, G. et. al., Rhodium(III) analogues of antitumour-active ruthenium(III) compounds: The crystal structure of [ImH][trans-RhCl4(Im)2] (Im=imidazole). Inorganica Chimica Acta 1998, 273 (1–2), 62-71. [5] Roddy, M.; Nelson, T.; Appledorn, D. M.; Groppi, V. CellPlayer 96-Well Kinetic Cell Migration and Invasion Assays; Essen BioScience: Ann Arbor, Michigan, 2013. [6] Levina, A.; et. al, Biotransformations of anticancer ruthenium(III) complexes: an X-ray absorption spectroscopic study. Chemistry 2013, 19 (11), 3609-19. [7] Van Wart, et. al., The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proceedings of the National Academy of Sciences 1990, 87 (14), 5578-5582. [8] Sava, G.et. al; Dual Action of NAMI-A in Inhibition of Solid Tumor Metastasis: Selective Targeting of Metastatic Cells and Binding to Collagen. Clinical Cancer Research 2003, 9 (5), 1898-1905.
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A series of Rh(III) complexes with biologically relevant ligands have been synthesised and evaluated for the anticancer properties; The Rh-imidazole complex (2) and Rh-indazole complex (3) displayed appreciable cytotoxicity and less resistance against selected cancer cell lines, and Rh-DMSO complex (1) possessed pronounced antimetastatic properties comparable to NAMI-A, the Ru-ancancer agent currently undergoing clinical trials; The antimetastatic effect of Rh complex 1 is most likely due to the ligand exchange reactions between 1 and the peripheral collagen network of metastatic tumours; The synchrotron XAS offers valuable biochemical information on the physiological transformation and speciation of metal-based anticancer compounds.
Acknowledgement This work is financial supported by the Australian Research Council and the Australian Synchrotron J. L. is grateful to Australian Synchrotron, Bosch Institute Molecular Biology Facility, Sydney Microscopy and Microanalysis Unit and School of Chemistry, The University of Sydney, for the instrumentation and technical support.
