Background
Cisplatin [cis-diamminedichloroplatinum(II)] is a platinum-based anticancer drug commonly used for treatment of different types of cancer, including ovarian, testicular, head and neck or lung carcinomas [
1]. Although cisplatin exerts significant anti-tumour activity, cisplatin-based therapies cause numerous side effects, such as nephrotoxicity, neurotoxicity and nausea. The intrinsic as well as acquired resistances to cisplatin-based therapy also represent substantial complications for the treatment of tumours.
For this reason, vast efforts were committed to develop novel platinum-based complexes to overcome platinum resistance, to reduce cisplatin side effects and to introduce novel mechanisms of anti-cancer action. Two derivatives, carboplatin (cis-diammine-(1,1-cyclobutanedicarboxylato)platinum(II)) and oxaliplatin (trans-R,R.cyclohexane-1,2-diammine)oxalatoplatinum(II)), have been introduced [
2]. Taken together, these three drugs represent all of the available platinum drugs approved by the Food and Drug Administration for clinical use [
3]. In addition to substantial side effects, these three drugs also require intravenous administration, therefore a new generation of platinum drugs represented by satraplatin (JM216) [(
OC-6-43)-bis(acetato)amminedichloro(cyclohexylamine)platinum(IV)] has been generated. JM216 represents the first orally administered platinum compound which has been evaluated in clinical trials [
2,
4,
5].
LA-12, [(
OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum (IV)], is a hydrophobic platinum(IV) complex structurally similar to JM216, which contains 1-adamantylamine instead of cyclohexylamine non-leaving ligand [
6] which provides this drug different properties. LA-12 has shown a higher cytotoxicity than JM216 when tested on a panel of 14 cancer cell lines of various origin and different cisplatin sensitivity [
6,
7] and no cross-resistance with cisplatin [
6,
8]. LA-12 has also displayed (i) higher antitumor activity in comparison with cisplatin and JM216, (ii) favourable pharmacokinetics and (iii) relatively low acute toxicity in a panel of pre-clinical
in vivo studies [
9‐
11].
The cytotoxic mode of action of cisplatin is mediated by its interaction with DNA to form DNA adducts, primarily intra-strand crosslink adducts, which activate several signal transduction pathways, including those involving ATR, p53, p73, and MAPK, and culminate in the activation of apoptosis [
12]. JM216 has a mechanism of action similar to that of cisplatin, inducing the formation of DNA adducts and inter- and intra-strand crosslinks and resulting in G2 arrest and induction of apoptosis [
13]. The principle mechanisms of LA-12 effects are not fully understood to date, although there is some evidence that exposure to LA-12 can disrupt cell proliferation and induce apoptosis more potently than cisplatin in both p53 dependent and independent manners [
14,
15]. Interestingly, LA-12 induces unique changes in the profile of gene expression compared to cisplatin, indicating a distinct mode of action resulting in the differential activation of both p53-dependent and p53-independent gene targets [
16].
Besides the well known cisplatin DNA-mediated effect, the other mechanism of cisplatin action was described demonstrating that cisplatin may also disrupt the function of some proteins, including heat shock protein 90 (Hsp90) [
17]. According to this study, cisplatin associates with the molecular chaperone Hsp90 and reduces its' chaperone activity. For example, this can result in the degradation of steroid receptors [
18] and/or inhibition of interaction between Hsp90 and inositol hexakisphosphate kinase-2 (IP6K2), which disrupts the inhibitory effect of Hsp90 on IP6K2, leading to increased diphosphoinositol pentakisphosphate (IP7) formation and sensitisation of cancer cells to apoptosis [
19,
20].
Since it is supposed that LA-12 undergoes metabolic changes and serves actually as a pro-drug for chemotherapeutically active platinum analogues, it is possible that these active metabolites can interact with proteins analogous to cisplatin, resulting in stronger cytotoxic or cytostatic effects. According to this hypothesis we have analyzed whether LA-12 can bind to Hsp90 resulting in inhibition of its protective chaperoning function such as folding, stabilization, activation and assembly of its "client" proteins.
Discussion
Platinum(IV) complexes represent a class of anticancer agents that are activated by reactions with reducing agents present in body liquids after their administration. Thus, the platinum(IV) complexes display potential advantages over their platinum(II) counterparts because of their greater stability and bioreductive activation, thereby allowing a greater proportion of the drug to arrive at the target intact. Thus, it is not surprising that the cellular uptake of LA-12 was faster and more effective than that of cisplatin (Figure
1).
Hsp90 is a molecular chaperone and is one of the most abundant proteins expressed in cells [
25]. Molecular chaperone Hsp90 is a member of the heat shock protein family, which is up-regulated in response to stress and its'
in vivo function is ATP-dependent [
26]. Hsp90 exists as a homodimer, which contains three domains. The N-terminal domain contains an ATP-binding site that binds the natural products geldanamycin and radicicol. Hsp90 function depends upon the ability of the N-terminal domain to bind and hydrolyze ATP, which is regulated and coupled with the conformational changes of the Hsp90 dimer [
27]. The middle domain exhibits high affinity for co-chaperones as well as client proteins [
28]. The structure of the C-terminus of Hsp90 contains the MEEVD sequence, which is known to bind co-chaperones that contain multiple copies of the tetratricopeptide repeat (TPR), a 34 amino acid sequence [
29]. Interestingly, the middle C-terminal part of Hsp90 has been implicated biochemically as the site of a putative second cryptic ATP-binding site on Hsp90 [
26]. The contribution of this site to the overall regulation of chaperone function is not clear, but novobiocin has been reported to interact with this site and alter the conformation of the chaperone [
30]. Importantly, cisplatin has also been found to bind to an Hsp90 site that overlaps the putative ATP/novobiocin binding site in this region, leading to inhibition of some Hsp90 activities [
17,
26]. The observed higher affinity of Hsp90 to LA-12 may be explained by very specific properties of C-terminal domain of Hsp90. This domain contains several solvent-exposed hydrophobic residues, which are responsible for binding of client protein such as unligated glucocorticoid receptor [
31]. Consequently the hydrophobic properties of LA-12 could explain its preferential binding to Hsp90 in contrast to cisplatin.
The ability of LA-12 to inhibit Hsp90 function was shown using several approaches. (i) EMSA, based on the fact that under physiological conditions the chaperone activity of Hsp90 is important for the transcriptional activity of genotypically wild-type p53 [
21] clearly demonstrated LA-12 as potent inhibitor of Hsp90 chaperoning function. (ii) Determination of p53 conformation in cells expressing various p53 mutants, which were exposed to LA-12, revealed that the unfolded mutant p53 proteins that do not rely on Hsp90 activity are less stable than folded mutant p53 proteins that require Hsp90 activity. These findings are in agreement with previous work showing that the half-life of p53 mutant positively correlates with their conformational stability [
22].
It was shown that inhibition of Hsp90 results in ubiquitination and degradation of primarily unfolded p53 mutants. This MDM2-independent degradation of unfolded p53 mutant is mediated by chaperone interacting protein CHIP. Our results show that CHIP is responsible for LA-12 mediated degradation in the same way as 17-AAG, which supports our theories finding LA-12 as a specific Hsp90 inhibitor.
Accordingly to both higher cellular uptake of LA-12 and at least 10-fold higher presence of platinum bound to Hsp90 in cells exposed to LA-12 compared to cells treated by cisplatin (Table
1), a stronger suppressive effect of LA-12 on Hsp90 activity and function in relation to cisplatin could be expected. This statement was confirmed by the ability of LA-12 to influence the stability and degradation rate of other Hsp90 client proteins, such as estrogen receptor and cyclin D1 compared to cisplatin, which did not decrease the level of these proteins.
Methods
Cell cultures
All human cancer cell lines were obtained from ATCC. Cell lines BT-474, MDA-MB-468, MDA-MB-231, T-47D, BT-549 and SK-BR-3 contain different p53 mutations (E285K, R273H, R280K, L194F, R249S and R175H, respectively), whereas the wild-type allele was lost in all these cell lines. MCF-7 cells contain wild-type p53, and H1299 cells are p53-null. Cells were periodically checked for morphology by microscope and regularly screened for mycoplasma using Hoechst staining.
Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen, Paisley, Scotland) supplemented with 10% fetal bovine serum (Invitrogen, Paisley, Scotland), 300 mg/l L-glutamine (Invitrogen, Paisley, Scotland), 100 U/ml penicillin (Invitrogen, Paisley, Scotland) and 0.1 mg/ml streptomycin (Invitrogen, Paisley, Scotland), in a humidified incubator at 37°C in 5% CO2 atmosphere. Cells were grown to 80% confluence prior to experimental treatments.
Chemicals
The following anti-neoplastic drugs were used in this study: LA-12, cisplatin (Pliva-Lachema a.s., Brno, Czech Republic), 17-AAG (Alexis Biochemicals, Lausen, Switzerland). LA-12 and cisplatin were dissolved in DMSO and diluted in cell medium to 0.5 mM stock solution and 17-AAG was dissolved in DMSO to 2 mM stock solution. Stock solutions of LA-12 and cisplatin were freshly prepared before use. The final concentration of DMSO in cell culture medium did not exceed 0.2%.
SDS-PAGE and western blotting
SDS-PAGE and western blotting were performed as described previously [
16]. The following antibodies were used: DO-1 mouse monoclonal antibody directed towards the epitope
20-SDLWKL-
25 within the p53 N-terminal region [
32], CM-1 rabbit polyclonal antibody against human p53 protein (in house antibody), CHIP-11.1 mouse monoclonal antibody recognizing full length as well as truncated CHIP protein (in house antibody), Estrogen Receptor (clone SP1) rabbit monoclonal antibody (Lab Vision, Fremont CA, USA), CD1 mouse monoclonal antibody recognizing Cyclin D1 (in house antibody), EEV1-2.1 mouse monoclonal antibody against Hsp90 protein (in house antibody) and AC-40 monoclonal antibody recognizing actin (Sigma-Aldrich Inc. St. Louis, USA), which was used as a protein loading control. Final concentration of all antibodies used for immunodetection of proteins was 1 μg/ml in 5% milk containing 0.1% Tween 20 in PBS.
Immunoprecipitation
Immunoprecipitation of conformationally different forms of p53 protein was performed using 1 μg of mouse monoclonal antibodies PAb1620 recognizing the wild-type conformation of human p53 protein at native form [
33,
34] and PAb240 recognizing the mutant conformations of p53 at native form [
35], as described previously [
33,
35]. For immunoprecipitation of Hsp90 protein for platinum amount measurement by AAS, cells were lysed in 150 mM NaCl, 50 mM HEPES (pH 7.2) and 0.5% Tween 20 and sonicated. Protein concentrations were measured using Bradford's assay. 200 μl of cell lysate containing 400 μg of total protein was incubated overnight together with 2 μg of EEV2-1.1 mouse monoclonal antibody against Hsp90β protein (in house antibody) and AC88 mouse monoclonal antibody against Hsp90 protein (Stressgen Biotech Corp., Victoria, BC, Canada). 15 μl of G-protein sepharose beads (GE Healthcare, Wien, Austria) was used to precipitate the antibody. After three washes in lysis buffer, beads were resuspended in 200 μl of 150 mM NaCl, 50 mM HEPES (pH 7.2) and 0.5% Tween 20 and boiled for 10 min.
Preparation of samples for AAS platinum amount measurement
Cells were incubated for the given time intervals with different doses of LA-12 and cisplatin. Control and treated cells were collected by centrifugation, washed three times with ice-cold PBS, lysed in 150 mM NaCl, 50 mM HEPES (pH 7.2) and 0.5% Tween 20 and sonicated. Protein concentrations were measured using Bradford's assay. 200 μl of cell lysate were used for platinum amount measurement by AAS.
In vitro p53 DNA Binding Assay
Human wild-type p53 and human Hsp90β were purified and activity of p53 was quantified by EMSA as described previously [
36]. Simplified, the assay is based on the ability of human recombinant Hsp90 to restore the specific DNA binding activity of human recombinant p53 at 37°C
in vitro. A preincubation with Hsp90 inhibitors prior to p53 inactivation at 37°C was carried out as described for geldanamycin [
21].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
VK designed, performed and analysed platinum accumulation experiments, performed immunoprecipitation experiments and prepared the manuscript, RH designed and performed degradation experiments and prepared the manuscript, DW performed EMSA experiments, EM performed CHIP transfection experiments, VH and DS performed AAS analysis, PM and PS contributed to the experimental design of the study, BV contributed to the conception and design of the entire study and the final editing of the manuscript. All authors read and approved the manuscript.