The central findings in the present study are: (a) GSE up-regulates p21 expression via ROS-mediated ERK1/2 activation in human colon carcinoma HT29 cells; (b) GSE does not affect transcriptional or post-translational mechanisms but modulates post-transcriptional and translational mechanisms for the up-regulation of p21 expression; (c) GSE-induced p21 level, in part, mediates GSE-induced G1 arrest; and (d) GSE-induced p21 expression moderately suppresses apoptosis during early time point, however, it is not a sustained action.
We observed a sustained activation of ERK1/2 as well as p21 up-regulation after GSE treatment of HT29 cells. Activation of ERK1/2 usually occurs in response to proliferative signals [32
]; however, it has been recognized that sustained activation of ERK1/2 can lead to cell cycle arrest [34
]. In our study with MEK inhibitors, we found that sustained activation of ERK1/2 is required for p21 up-regulation through some yet unknown mechanisms, as blockade of ERK1/2 by chemical inhibitors almost completely abrogated the induction in p21 levels by GSE. Next, we investigated the mechanism of GSE-induced activation of ERK1/2. There are reports that oxidative stress can induce MAPK signaling [36
]. Therefore, we anticipated that changes in intracellular redox conditions by GSE could be pertinent to MAPK activation. As anticipated, the pre-treatment of cells with NAC, a precursor of reduced glutathione, completely abrogated the activation of ERK1/2 as well as up-regulation of p21 levels. This is supported by our previous study wherein GSE was found to enhance ROS levels in prostate cancer cells [37
]. Therefore, it is likely that ROS production by GSE might be resulting in oxidative stress in cells. We also observed a decreased level of intracellular reduced glutathione upon GSE treatment in HT29 cells; this observation further supports the generation of intracellular oxidative stress by GSE. Another study has reported the involvement of ROS-dependent mechanism in p21 up regulation in which diethylmaleate induced p21 mRNA levels in Hela cells by p53-independent but oxidative stress-mediated mechanisms [38
]. Diethylmaleate creates oxidative stress by depleting intracellular glutathione levels. Since, NAC pre-treatment abrogated GSE-induced activation of ERK1/2; there is also a possibility that GSE-induced oxidative stress might inactivate MAPK specific phosphatases resulting in sustained activation of ERK1/2. However, additional studies are required to explore the existence of this possibility.
P21 is an endogenous CDKI with broad specificity for CDKs [39
]. In addition to its prominent role in affecting cell cycle arrest, it is also involved in multiple other cellular processes such as apoptosis, senescence and differentiation [40
]. The cellular levels of p21 can be regulated by different mechanisms involving both transcriptional and post-transcriptional mechanisms [11
]. In the present study, a modest increase in p21 mRNA levels was observed by GSE which must be occurring through a p53-independent mechanism, as HT29 cells carry mutation in p53 gene. Further, GSE treatment showed a marginal but non significant increase in the promoter activity of p21. These observations suggested that up-regulation of p21 by GSE does not occur at transcriptional level; therefore, the possibility of involvement of post-transcriptional/ translational mechanisms was also explored.
Post-translational mechanisms such as ubiquitination or phosphorylation followed by proteasomal degradation play an important role in controlling the turn over rate of many proteins [43
]. In case of p21, ubiquitination followed by proteasomal degradation is one such mechanism affecting the overall stability of this protein [46
]. Inhibition of this pathway results in increased stability of the p21.To study the involvement of such mechanisms in GSE-induced up-regulation in p21 protein levels; we conducted CHX chase experiment, in which GSE showed a marginal effect on the half-life of p21. These observations suggest that post translational mechanisms do not contribute significantly in increasing the stability/levels of p21 protein by GSE.
In addition to transcriptional and post-translational mechanisms, the post-transcriptional mechanisms regulating the levels of p21 have been observed under different experimental conditions. The message for p21 has been shown to be stabilized through binding of either HuR or poly(C)-binding protein at 3’ UTR region in human MDA-MB468 breast cancer cells upon EGF treatment [47
]. Similarly, post-transcriptional modifications were involved in increasing the stability of mRNA in short wavelength UVC-induced and p53-dependent up-regulation of p21 protein [48
]. To study the involvement of such mechanisms, we conducted actinomycin D chase experiment. Our results showed that the levels of p21 protein, which indirectly represents the mRNA levels were relatively higher in comparison to untreated cells at all the time points studied, thereby indirectly implying that there is an increased stabilization of the p21 mRNA in GSE-treated cells. Thus, GSE may be stabilizing p21 mRNA levels by post-transcriptional mechanisms with concomitant increase in its mRNA levels with a resultant increase in translation efficiency leading to overall increase in p21 protein levels.
In an effort to explore the biological implications of robust up-regulation of p21 levels by GSE, HT29 cells were stably knocked-down for p21, and cell cycle phase distribution and apoptosis were analyzed. Knock-down of p21 resulted in abrogation of primarily G1 arrest with no impact on transient G2/M arrest caused by GSE treatment. This suggests that up-regulation of p21 by GSE is primarily involved in causing arrest at G1 phase. Additionally, knock-down of p21 resulted in marginal increase in apoptosis by GSE at early time point (12 h), and this effect diminished by 24 h of GSE treatment, suggesting that p21 does not play any major role in GSE-induced apoptosis of HT29 cells. It has been reported that the cellular response switches from cell cycle arrest to apoptosis in event of selective degradation or transcriptional repression of p21 [49
]. However, in the present study, GSE increased both apoptosis as well as p21 level, and knockdown of p21 has marginal effect on GSE-induced apoptosis. Thus, further studies are needed to identify the molecule/s targeted by GSE for apoptosis induction in HT29 cells.
Taken together, these results demonstrate that GSE causes oxidative stress in HT29 cells that mediates the activation of MEK/ERK pathway for up-regulation of p21 levels. Up-regulation in p21 levels by GSE involves post-transcriptional stabilization of p21 mRNA with concomitant increase in translation of the message. GSE-induced p21 level is required for G1 arrest and has only marginal effect on apoptosis caused by GSE in HT29 cells. Thus, the findings of this study suggest that GSE could be a potential chemopreventive agent due to its ability to regulate the growth of colorectal cancer HT29 cells via targeting of p21/Cip1, a critical regulator of cell cycle progression.