Identification of cisplatin-binding proteins using agarose conjugates of platinum compounds

Abstract

Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94), heat shock protein 90 (HSP90), calreticulin, valosin containing protein (VCP), and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment.

Figure 1. Structures of cisplatin and carboplatin.

Figure 2. Synthesis of Pt complexes 3 and 5.

Figure 3. ¹H NMR of Pt complexes.

¹H NMR spectra in D₂O shows methylene and methine proton shifts in complex 5 as compared to that of 3. ¹⁵N-labeled (top) and non-labeled (middle) complex 5 exhibit similar chemical shifts and splitting patterns.

Figure 4. ¹⁹⁵PtNMR spectra of complexes 3 and 5 in D₂O.

Pt complex 5 shows an upfield chemical shift as compared to that of 3. Similar chemical shifts are observed for the ¹⁵N-labeled (top) and non-labeled (middle) complex 5.

Figure 5. HR ESI positive mode MS spectrum of complex 5.

Figure 6. Cisplatin-binding protein pull-down assay results.

(A) Pull-down assay samples using HEI-OC1 cells. Agarose without Pt conjugation (No Pt) was used as a control. 2NH₃Pt-agarose pulled down several proteins in significant amounts, while 2ClPt-agarose only pulled down fewer proteins in lower amounts. The total cell lysate (Lysate) sample before pull-down is to show relative expression levels of proteins with different molecular mass. (B) 2NH₃Pt-agarose pull-down assay results. Several protein bands with high intensity appeared in all cells tested, with some minor band differences in different lanes. Gel bands were excised from HEI-OC1 samples and analyzed by mass spectrometry. Protein names identified in gel regions a, b, c, d and e are shown in Table 1.

Figure 7. Protein expression and cisplatin-binding of HSP90α and HSP90β.

(A) HSP90 protein expression analysis. Total cell lysates from cell lines used for pull-down assay were analyzed by Western blotting, using antibodies for HSP90α and HSP90β. KPT11 cell sample showed very low expression of HSP90α. (B) Pt-agarose pull-down assay using 293T cells transfected with FLAG-HSP90α or -HSP90β. 2NH₃Pt-agarose pulled down the two HSP90 isoforms with similar amounts.

Figure 8. HSP90α expression and cell viability.

(A) MTT assay on cells treated with cisplatin for 2 days showed that kidney proximal tubule cells (KPT11 and KPT2) had lower viability compared to kidney distal tubule KDT3 cells (**P<0.01 between either one of proximal tubule cell lines and KDT3). (B) Western blotting confirmed that KPT2-HSP90α cell lines (HSP90α#1 and #2) express higher levels of HSP90α compared to parental KPT2 or KPT2 with empty pBabe vector control. (C) Cellular growth for 2 days was assessed by MTT assay, and KPT2-HSP90α cell lines showed faster cellular growth compared to KPT2-pBabe cell lines (**P<0.01). (D) Cisplatin treatment for 2 days showed that KPT2-HSP90α cell lines had lower viability compared to KPT2-pBabe cell lines (**P<0.01 between either one of KPT2-HSP90α and either one of KPT2-pBabe).