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, 14 (3), 241-52

Cyclin A2 and CDK2 as Novel Targets of Aspirin and Salicylic Acid: A Potential Role in Cancer Prevention

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Cyclin A2 and CDK2 as Novel Targets of Aspirin and Salicylic Acid: A Potential Role in Cancer Prevention

Rakesh Dachineni et al. Mol Cancer Res.

Abstract

Data emerging from the past 10 years have consolidated the rationale for investigating the use of aspirin as a chemopreventive agent; however, the mechanisms leading to its anticancer effects are still being elucidated. We hypothesized that aspirin's chemopreventive actions may involve cell-cycle regulation through modulation of the levels or activity of cyclin A2/cyclin-dependent kinase-2 (CDK2). In this study, HT-29 and other diverse panel of cancer cells were used to demonstrate that both aspirin and its primary metabolite, salicylic acid, decreased cyclin A2 (CCNA2) and CDK2 protein and mRNA levels. The downregulatory effect of either drugs on cyclin A2 levels was prevented by pretreatment with lactacystin, an inhibitor of proteasomes, suggesting the involvement of 26S proteasomes. In-vitro kinase assays showed that lysates from cells treated with salicylic acid had lower levels of CDK2 activity. Importantly, three independent experiments revealed that salicylic acid directly binds to CDK2. First, inclusion of salicylic acid in naïve cell lysates, or in recombinant CDK2 preparations, increased the ability of the anti-CDK2 antibody to immunoprecipitate CDK2, suggesting that salicylic acid may directly bind and alter its conformation. Second, in 8-anilino-1-naphthalene-sulfonate (ANS)-CDK2 fluorescence assays, preincubation of CDK2 with salicylic acid dose-dependently quenched the fluorescence due to ANS. Third, computational analysis using molecular docking studies identified Asp145 and Lys33 as the potential sites of salicylic acid interactions with CDK2. These results demonstrate that aspirin and salicylic acid downregulate cyclin A2/CDK2 proteins in multiple cancer cell lines, suggesting a novel target and mechanism of action in chemoprevention.

Implications: Biochemical and structural studies indicate that the antiproliferative actions of aspirin are mediated through cyclin A2/CDK2.

Figures

Figure 1
Figure 1
Aspirin and salicylic acid down-regulate cyclin A2 protein levels in multiple cell lines. A and B respectively represent the effect of aspirin and salicylic acid on cyclin A2 protein levels in HT-29 cells. C and D represent comparison of the effect of aspirin and salicylic acid in multiple cancer cell lines. For C and D, the intensity of bands in various western blots were quantified and expressed as percentage of control. P-value < 0.001‡, <0.01†
Figure 2
Figure 2
Down-regulation of cyclin A2 by aspirin and salicylic acid is mediated by 26S proteasomal pathway. A, effect of lactacystin on the ability of aspirin and salicylic acid to decrease cyclin A2 protein levels in HT-29 cells. B, represents the quantification of the bands in blot A. C and D, aspirin and salicylic acid down-regulate exogenously expressed DDK-tagged cyclin A2 protein. C and D, respectively represent immunoblots probed with anti-DDK tagged antibody and anti-cyclin A2 antibody. Positions of the exogenous and endogenous cyclin A2 were shown by arrows (see the text for details).
Figure 3
Figure 3
Aspirin (A) and salicylic acid (C) decrease cyclin A2 mRNA levels in a concentration dependent fashion. B and D respectively represents, the ethidium bromide stained ribosomal RNA pattern of blots in A and C (see the text for details).
Figure 4
Figure 4
Aspirin/salicylic acid down-regulate CDK2 protein/mRNA levels and activity. A, aspirin and salicylic acid down-regulate CDK2 protein in HT-29 cells. B, quantification of the band in blot A, expressed as percentage control. C, Northern blot analysis of CDK2 in response to salicylic acid treatment in HT-29 cells. D, ethidium bromide stained ribosomal RNA pattern of blot C. E and F, respectively represent CDK2 activity at two different concentrations (0.5 mM and 1.5 mM) in HT-29 and SK-MEL-28 cells, numbers on the blot represent intensities expressed as percentage of control. The lower panel shows the Coomassie blue stained histones following electrophoresis (see the text for details).
Figure 5
Figure 5
The inclusion of salicylic acid increases the ability of anti-CDK2 antibody to immunoprecipitate CDK2 from naïve cell lysates and recombinant CDK2 protein. A, anti-cyclin A2 antibody (cat. Number ab32386; Abcam) immunoblots of anti-CDK2 immunoprecipitates (cat. Number-05-596, EMD Millipore), showing the presence of increased levels of cyclin A2 with increased concentration of salicylic acid. In this blot, the area that has cyclin A2 bands are only shown. B, anti-CDK2 antibody immunoblots of the anti-CDK2 immunoprecipitates, shows the presence of increased levels of CDK2 with increased concentrations of salicylic acid. C, shows the results of the in-vitro kinase assay performed on anti-CDK2 immunoprecipitates in an experiment similar to figure 5A. For the experiment in 5C, salicylic acid was pre-incubated with lysate before immunoprecipitation, but not included in the kinase assay. D, shows the results of in-vitro kinase assay performed on anti-CDK2 immunoprecipitates. For the experiments in Fig. 5D, lysates were not pre-incubated with salicylic acid before immunoprecipitation, but was included during the kinase assay. E, anti-CDK2 immunoblot of anti-CDK2 immunoprecipitate of recombinant CDK2, immunoprecipitation was carried out in the presence of increasing concentration of salicylic acid. Ig-H, immunoglobulin heavy chain, Ig-L immunoglobulin light chain.
Figure 6
Figure 6
A, effect of pre-incubation of salicylic acid with CDK2 on fluorescence due to ANS. CDK2 (1.6 μM) was incubated with ANS (50 μM) alone or with salicylic acid at different concentrations, fluorescence measured as described in the text. Salicylic acid mediated decrease in fluorescence was compared with fluorescence due to ANS/CDK2. The decrease in fluorescence was expressed as a percentage of control; B, is the molecular docking studies showing interactions of salicylic acid with CDK2; C, a model showing potential salicylic acid binding to CDK2. We predict that salicylic acid binds to an allosteric site on CDK2, similar to a site described for ANS binding to CDK2. Binding of salicylic acid to CDK2 changes the conformation; increases the ability of anti-CDK2 antibody to immunoprecipitate CDK2 due to a better exposure of the epitope. Binding of salicylic acid to CDK2 would also quench the fluorescence due to ANS. We predict that potential allosteric inhibitors could be developed by screening new salicylic acid derivatives with allosteric binding potential and inhibition of CDK2 activity.

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