Comparison of click-capable oxaliplatin and cisplatin derivatives to better understand Pt(ii)-induced nucleolar stress

Abstract

Pt(ii) chemotherapeutic complexes have been used as predominant anticancer drugs for nearly fifty years. Currently there are three FDA-approved chemotherapeutic Pt(ii) complexes: cisplatin, carboplatin, and oxaliplatin. Until recently, it was believed that all three complexes induced cellular apoptosis through the DNA damage response pathway. Studies within the last decade, however, suggest that oxaliplatin may instead induce cell death through a unique nucleolar stress pathway. Pt(ii)-induced nucleolar stress is not well understood and further investigation of this pathway may provide both basic knowledge about nucleolar stress as well as insight for more tunable Pt(ii) chemotherapeutics. Through a previous structure-function analysis, it was determined that nucleolar stress induction is highly sensitive to modifications at the 4-position of the 1,2-diaminocyclohexane (DACH) ring of oxaliplatin. Specifically, more flexible and less rigid substituents (methyl, ethyl, propyl) induce nucleolar stress, while more rigid and bulkier substituents (isopropyl, acetamide) do not. These findings suggest that a click-capable functional group can be installed at the 4-position of the DACH ring while still inducing nucleolar stress. Herein, we report novel click-capable azide-modified oxaliplatin mimics that cause nucleolar stress. Through NPM1 relocalization, fibrillarin redistribution, and γH2AX studies, key differences have been identified between previously studied click-capable cisplatin mimics and these novel click-capable oxaliplatin mimics. These complexes provide new tools to identify cellular targets and localization through post-treatment Cu-catalyzed azide-alkyne cycloaddition and may help to better understand Pt(ii)-induced nucleolar stress. To our knowledge, these are the first reported oxaliplatin mimics to include an azide handle, and cis-[(1R,2R,4S) 4-methylazido-1,2-cyclohexanediamine]dichlorido platinum(ii) is the first azide-functionalized oxaliplatin derivative to induce nucleolar stress.

Fig. 1. Azide-functionalized cisplatin and oxaliplatin mimics used in this study.

Fig. 2. Synthesis of 2.

Fig. 3. X-ray structures of the l-(+)-tartrate salt of the enantiomerically pure DACH ligand found in 2 and [Pt(DACH-CH₂N₃)I₂], the diiodo analogue of 2.

Fig. 4. dPAGE analysis of Pt(ii) complexes incubated with HP DNA. Complexes labeled as “click” were first incubated with HP for 24 h followed by a click reaction with Alexa-647 DBCO for an additional 24 h, giving a total DNA incubation time of 48 h. Control complexes were incubated for 48 h with HP with no click reaction. All samples were purified by spin column before analysis by dPAGE. Grey: DNA stained with SYBR gold (539 nm emission wavelength), red: DNA-Pt-647 DBCO complex (671 emission wavelength).

Fig. 5. Confocal images of fluorescent cellular localization of azide-modified Pt(ii) complexes. Panel (A) shows fluorescent labeling of A549 cells treated with 50 μM of azide-modified Pt(ii) for 24 h and subsequent CuAAC with alkyne-tagged Alexa-488. The Pt(ii)-free and Cu-free controls show little to no signal indicating low background fluorescence. Panel (B) shows the nuclei labeled via DAPI stain. Panel (C) shows the overlay of Alexa-488 (green) labeling and DAPI (magenta).

Fig. 6. Visualization and quantification of NPM1 relocalization (NPM1: green, DAPI: gray) induced by 1,3-Pt and azido-Pt. Treatment conditions indicated (10 or 20 μM for Pt(ii) complexes, 5 nM for ActD) in A549 cells at 24 h treatment. Representative images and CV calculations based on 3 trials are shown. Full cell images found in ESI, Fig. S2. For each treatment data set, boxes represent median, first and third quartiles, and vertical lines are the range of data with outliers (see Experimental Section). Scale bar = 10 μm.

Fig. 7. Visualization and quantification of NPM1 relocalization (NPM1: green, DAPI: gray) induced by 1, 2, and 3. Treatment conditions indicated (10 or 20 μM for Pt(ii) complexes, 5 nM for ActD) in A549 cells at 24 h treatment. Representative images and CV calculations based on 3 trials are shown. Full cell images found in ESI, Fig. S4. For each treatment data set, boxes represent median, first and third quartiles, and vertical lines are the range of data with outliers (see Experimental Section). Scale bar = 10 μm.

Fig. 8. Nucleolar morphological changes monitored by fibrillarin (fibrillarin: green, DAPI: gray). Treatment condition at 10 μM for azido-Pt, 1,3-Pt, 2, and 3 and 5 nM for ActD in A549 cells at 24 h treatment. Nucleolar cap formation is indicated by a white arrow. Scale bar = 10 μm.

Fig. 9. Visualization and quantification of γH2AX (γH2AX: green, DAPI: gray) as an indicator of DDR induced by azido-Pt and 1,3-Pt. Treatment conditions indicated (10 μM for Pt(ii) complexes, 5 nM for ActD) in A549 cells at 24 h treatment; average of 3 trials and std; quantifications performed described in Experimental Section. Scale bar = 10 μm. ***p < 0.05, N.S. p > 0.05.

Fig. 10. Visualization and quantification of γH2AX (γH2AX: green, DAPI: gray) as an indicator of DDR induced by 2 and 3. Treatment conditions indicated (10 μM for Pt(ii) complexes, 5nM for ActD) in A549 cells at 24 h treatment; average of 3 trials and std; quantification performed described in Experimental Section. Scale bar = 10 μm. ***p < 0.05, N.S. p > 0.05.