PEITC causes rapid depletion of mutant p53 by a post-transcriptional mechanism
Treatment of human H596 (G245C) non-small cell lung cancer cells with 15 µM PEITC caused a rapid depletion of mutant p53 protein. Depletion occurred as early as 2 h and peaked at 4 h; it was sustained during 24 h treatment and was concentration-dependent (). The expression of other proteins, including Bax, DR5, Hsp90, and JNK, was not altered (not shown). To determine whether depletion occurs at the post-transcriptional or transcriptional level, reverse transcription-PCR was performed on RNA derived from DMSO and PEITC-treated H596 cells. As shown in , 2, 4 and 6 h after PEITC treatment, the mutant p53 mRNA levels remained unchanged. Therefore, PEITC depleted mutant p53 without causing changes in the p53 mRNA expression levels. These results strongly suggest that PEITC depletes mutant p53 at the post-transcriptional level. We next examined the effects of other widely studied naturally-occurring ITCs, benzyl ITC (BITC), SFN and AITC, on mutant p53 protein levels. H596 cells were incubated with PEITC, BITC, SFN or AITC for 2 h. shows that BITC at 10 µM significantly reduced the level of mutant p53, while at 20 µM it showed a similar potency as PEITC. However, neither SFN nor AITC significantly affected mutant p53 protein levels, even at 40 µM; although at 60 µM SFN slightly reduced mutant p53 levels. To further investigate the structural requirement of ITCs to deplete mutant p53, N-methylphenethylamine (NMPEA), an analog of PEITC lacking an ITC functional group was used. shows that NMPEA at 15 µM failed to alter the mutant p53 protein level even up to 24 h or at 60 µM for 2 h. Because the ITC functional group is highly electrophilic, these results indicate that the ITC group is essential for the effect, possibly through binding to target proteins. Moreover, the structure of the side-chain moiety appears to dictate the potency of mutant p53 depletion by ITCs.
PEITC causes rapid depletion of mutant p53 in H596 cells
PEITC depletes mutant p53 in a variety of cancer cells
We next examined whether the depletion of mutant p53 by PEITC is cell type-specific. Various tumor cells with mutant p53 were used, including MDA-MB-231 (breast cancer, R280K), MDA-MB-468 (breast cancer, R273H), DU145 (prostate cancer, P274L/V223F), SW480 (colon cancer, R273H) and SCC-4 (oral cancer, P151S). Notably, while the types of mutations among the cell lines vary, all are located in the core DNA-binding domain. The five cell lines were treated with 10 µM or 15 µM PEITC, depending on the observed cytotoxicity. shows that PEITC depleted mutant p53 in all five cell lines in a time-dependent manner, and certain cells appeared more responsive than others. These results clearly show that the effects of PEITC on mutant p53 are not specific to a certain p53 mutation.
PEITC depletes p53 protein in human cancer cells with different p53 mutation, but not in cells with wild type p53
Wild type p53 is not a target for depletion by PEITC in cancer cells
To determine whether wild type p53 is affected by PEITC treatment, we used A549 lung cancer cells, HCT116 colon cancer cells, MCF-7 breast cancer cells and MCF-10A human normal mammary epithelial cells, all expressing wild type p53. Cell lines were treated with 15 or 20 µM PEITC for 2, 4, 8 and 24 h. Contrary to mutant p53, wild type p53 expression did not decrease over the treatment period. In fact, it appeared marginally elevated at 24 h in MCF-7 cells (), suggesting that PEITC may moderately activate p53 in cells with wild type p53. To investigate whether the conformation of p53 contributes to the differential response to PEITC, we used H1299 lung cancer cells (null p53) stably transfected with temperature-sensitive p53 mutant 143Ala (H1299-143A) (13
). p53 mutant 143Ala can have two conformations: at 32.5°C it possesses a strong DNA-binding ability and generates transcriptional activity, thus, functioning like wild type p53, and at 37°C its ability to bind DNA and activate transcription is severely diminished or lost, acting as mutant p53. After treatment of H1299-143A cells with PEITC for 24 h, p53 mutant 143A protein was depleted more robustly at 37°C than at 32.5°C (). To rule out the possibility that lower temperature caused the effect, H1299 cells stably expressing non-temperature-sensitive p53 mutant R175H (H1299-175H) were treated with PEITC for 24 h; the same extent of depletion was observed at both temperature (). Therefore, our data suggest that the differential response between mutant and wild type p53 to PEITC is likely due to the difference in their conformation.
SARs for the depletion of mutant p53 by ITCs
BITC and PEITC are arylalkyl ITCs of different alkyl chain length with relatively high lipophilicities, whereas SFN, an alkyl ITC, is considerably more water-soluble. Previously, we showed BITC, PEITC and SFN exhibit differential activity toward total protein binding and apoptosis induction, following an order of BITC>PEITC>SFN (2
). Moreover, BITC and PEITC can modify proteins and tubulin to a greater extent than SFN, and the degree of binding correlates with the potency of the ITC to induce mitosis arrest and apoptosis (2
). Importantly, the same order of potency was seen toward mutant p53 depletion. NMPEA, an analog of PEITC without the ITC functional group, is completely devoid of all of these activities. Here, we studied the structural features of the ITC side chain moiety with relation to the extent of depletion of mutant p53. Compounds of more than one aromatic ring with greater lipophilicity than BITC and PEITC were used. These compounds are 2,2-diphenylethyl ITC and 4-phenoxybenzyl ITC. We also examined the effects of an electron-withdrawing and an electron-releasing substituent at the para position of the aromatic ring by using 4-chlorobenzyl ITC and 4-methoxybenzyl ITC. To investigate whether covalent binding by ITCs is a trigger of mutant p53 depletion, we selected ITCs with bulky substituent(s) adjacent to the –N=C=S to create steric hindrance, including D-α-methylbenzyl ITC and trityl ITC. Aromatic ITCs with varying alkyl chain length from phenyl ITC to 6-phenylhexyl ITC (6-PHITC) were used. Structures of the ITCs are shown in . The aliphatic compounds used include cyclohexylmethyl ITC, SFN, erucin and AITC.
Structure-activity relationships for the depletion of mutant p53 by ITCs
As shown in , mutant p53 is depleted similarly in H596 () and MDA-MB-231 () cancer cells treated with 10 or 20 µM ITCs, respectively, for 24 h. Also, at equimolar concentrations BITC and PEITC deplete mutant p53 to approximately the same extent, while SFN depletes p53 significantly less in H596 or MD-MBA-231 cells, compared to DMSO control. The presence of a phenyl ring appears important as its electron-withdrawing ability reduces the electron density around the carbon atom of the –N=C=S functional group rendering it more electrophilic (14
). The aliphatic ITCs, cyclohexylmethyl ITC, SFN, erucin and AITC, all have little to no mutant p53 depleting activity, whereas 2,2-diphenylethyl ITC and 4-phenoxybenzyl ITC deplete mutant p53 to an even greater extent than BITC (). Thus, increasing the lipophilicity of the side chain moiety by the addition of aromatic groups increases the activity of the ITC to deplete mutant p53. Introducing an electron-withdrawing or releasing substituent at the para position dose not exert much effect, as no significant difference was seen with 4-chlorobenzyl ITC and 4-methoxybenzyl ITC when compared with BITC. In MDA-MB-231 cells, 4-chlorobenzyl ITC may deplete to a greater extent than BITC; however, 4-methoxybenzyl ITC is just as efficient as BITC, if not more. D-α-methylbenzyl ITC and trityl ITC deplete little to no mutant p53; these ITCs have bulky substituents at the α-carbon adjacent to the ITC functional group. These differ from 2,2-diphenylethyl ITC, which does not have its bulky substituents on the α-carbon. Bulky substituents adjacent to the –N=C=S group could hinder binding of the ITC to proteins. The alkyl chain length also seems to influence the activity (). PITC does not deplete mutant p53, whereas BITC, PEITC, 3-PPITC, 4-PBITC, 5-PPeITC and 6-PHITC all deplete mutant p53 to different degrees. Interestingly, depletion is greatest when one, two or four methylene groups are inserted between the phenyl ring and the –N=C=S functional group (BITC, PEITC and 4-PBITC, respectively), and it is lowest when three methylene groups are present (3-PPITC). The large contrast of PPITC compared with PEITC and PBITC is notable.
Covalent modification of mutant (G245C) p53 DBD by ITCs and subsequent conformational changes
As in the case of tubulin degradation, it is possible that mutant p53 protein is depleted by ITCs as a result of direct binding to its cysteine residues and subsequent conformational changes. We used a monochlorobimane fluorometric assay (15
) to study cysteine modification of mutant (G245C) p53 DBD by ITCs, and compared intrinsic tryptophan fluorescence intensities as an indicator of conformational changes (16
). The G245C point mutation is especially relevant because it is the p53 mutation present in the H596 non-small cell lung cancer cell line studied here. shows the relative amount of free thiols present, as indicated by relative fluorescence intensities, in the mutant (G245C) p53 DBD after 1 h incubation with DMSO (negative control), ITCs or iodoacetamide (positive control). BITC, 2,2-diphenylethyl ITC, 4-methoxybenzyl ITC, 4-chlorobenzyl ITC, PEITC, and 3-PPITC show greater binding affinities for mutant (G245C) p53 DBD than do trityl ITC, cyclohexylmethyl ITC, SFN, AITC and PITC (p<0.05). These affinities correlate well with their respective activity to deplete mutant p53. However, the binding affinities are not always in agreement with the depleting potencies of ITCs. For example, 4-phenoxybenzyl ITC was one of the most effective depletors, but it did not display stronger binding to mutant p53 than the weaker depletors, such as D-α-methylbenzyl ITC. These results suggest that binding affinity to mutant p53 can only partially explain depleting activities and other factor(s) may be involved.
Cysteine binding in mutant (G245C) p53 DNA-binding domain by ITCs and subsequent conformational changes
Using intrinsic tryptophan fluorescence emission spectroscopy, we demonstrated that the conformation of mutant p53 changes to different degrees after its reaction with BITC, 4-phenoxybenzyl ITC, D-α-methylbenzyl ITC and SFN (). These ITCs were chosen because their binding affinities correlate either well or poorly with depletion. 4-Phenoxybenzyl ITC, a potent depletor, induces the greatest conformational change, despite its lower binding affinity for mutant p53 protein. BITC causes the second largest conformational change, followed by D-α-methylbenzyl ITC and finally SFN. D-α-methylbenzyl ITC shows a lower binding affinity than BITC (p<0.05), but a higher binding affinity than 4-phenoxybenzyl ITC (p<0.05); however, D-α-methylbenzyl ITC does not deplete mutant p53 to the same degree as 4-phenoxybenzyl ITC. The fact that D-α-methylbenzyl ITC induces less mutant p53 protein conformational change than 4-phenoxybenzyl ITC may explain why it does not deplete as efficiently. Similarly, despite its binding to mutant p53, SFN causes the least conformational change among ITCs tested, which correlates with its lack of depleting activity.
Mutant p53 cells are more sensitive to apoptosis induced by PEITC than wild type cells
The role of p53 in the induction of apoptosis by ITCs is not well established and likely to be cell-specific. Mutant p53 renders cancer cells resistance to chemotherapeutic drugs; if ITCs can deplete mutant p53, then ITCs should be more cytotoxic in cells with mutant p53 protein. To examine this possibility, we assessed the cytotoxicity induced by PEITC in cancer cells containing either wild type or mutant p53. Three sets of cell lines from lung, breast, and colon were used: A549 (wild type p53) and H596 (mutant p53) lung cancer cells, MCF-7 (wild type p53) and MDA-MB-231 and MDA-MB-468 (mutant p53) breast cancer cells, HCT116 (wild type p53) and SW480 (mutant p53) colon cancer cells. After treating with PEITC for 24 h, apoptosis was determined by a caspase-3 activity assay. As shown in , all the mutant p53 cancer cells appeared significantly more sensitive than the wild type cells to PEITC-induced apoptosis.
Cells with mutant p53 are more sensitive to PEITC-induced apoptosis than wild type cells, and induction of apoptosis by various ITCs in a cell line with mutant p53
Induction of apoptosis by various ITCs in a mutant p53 cell line
To examine the correlation between depletion of mutant p53 and apoptosis by ITCs, FITC-annexin V staining assay was used to quantify apoptotic cells, we treated MDA-MB-231 cancer cells with DMSO or 10 µM of various ITCs for 24 h, shows that treatment with BITC, 2,2-diphenylethyl ITC, 3-PPITC or 4-PBITC, which all deplete mutant p53 (), results in a greater induction of apoptosis than treatment with DMSO (p<0.05). 2,2-diphenylethyl ITC, the most potent depletors of mutant p53 studied, is the strongest inducer of apoptosis (p<0.05 compared to BITC). The percentage of apoptotic cells induced by trityl ITC, cyclohexylmethyl ITC or SFN, all of which deplete little to no mutant p53, does not differ from control. However, the correlation is not consistently evident. For example, 3-PPITC, which does not deplete mutant p53 to the same extent as BITC or 4-PBITC, induces the same level of apoptosis as these ITCs.