Resistance of colorectal cancer to established treatment regimens remains a major concern in oncology; thus attempts at improving the survival of patients affected by this disease depend largely on strategies targeting tumor cell resistance, which cannot be rationally planned without a detailed knowledge of the mechanisms underlying this phenomenon. A current paradigm regarding cancer chemotherapy indicates disabling of the intrinsic apoptotic pathway as a key factor in the response of tumor cells to anticancer drugs [
3,
5,
12]. Therefore, strategies aiming at re-establishing the cell's capability to activate a cell death program are an area of active research. The present study was performed in order to define the role of the p53/p21 pathway in the response of colorectal carcinoma cells to DOX, a cytotoxic agent that is typically devoid of effects in this tumor type. The results obtained in our cytotoxicity studies indicate that in the cell lines examined
p53 status is not unequivocally related to the response to DOX: in fact, while
p53-deficient cells (HT-29, HCT116-E6) are indeed less responsive than the
p53/wt parental HCT116 cell line, the highest resistance index was obtained for HCT116 p21-/- cells, harboring two wildtype
p53 alleles. As expected, treatment with DOX leads to p53 upregulation in the cell lines expressing wildtype p53; this effect has been thoroughly documented in colon cancer cells as well as in tumor cell lines derived from other tissues, and has been attributed to phosphorylation and subsequent stabilization of p53, possibly through activation of DNA-dependent protein kinase or ATM (ataxia-teleangectasia mutated) kinase (see e.g. [
20‐
22]). In HCT116 cells, p21 expression parallels p53 activation; however, data obtained in HT29 and HCT116-E6 cells clearly indicate the existence of p53-independent pathways for p21 induction, that have been extensively characterized (for a review see [
23]) and can be activated to variable extents (HCT116 E6 > HT29) upon exposure to DOX. Interestingly, the extent of the cytotoxic effects observed in the small panel of colon cell lines tested rather seems to parallel the cells' ability to upregulate p21 (HCT116 > HCT116-E6 > HT29 > HCT116 p21-/-). This result is somewhat unexpected: in fact, whereas the function of p21 in cell growth arrest following DNA damage has been established for a long time [
24], the role played by this protein in the ultimate fate of tumor cells exposed to cytotoxic agents is far from clear-cut [
14,
25]. In a number of studies, p21 has actually been reported to protect tumor cells against cell death induced by enforced p53 expression [
26] or by low doses of cytotoxic agents [
13,
27‐
30]. However, in other experimental settings, p53-dependent or -independent induction of p21 expression seems to be a prerequisite for apoptosis [
31‐
34] and to sensitize tumor cells to the action of different agents [
35‐
37]. The putative mechanisms by which p21 might actually induce apoptosis have recently been reviewed [
38], but still await full elucidation.
Interestingly, the situation outlined by our results does not seem to conform to either view: in fact, while induction of p21 in p53-proficient and -deficient cell lines is associated with increased response to drug treatment, this was not accompanied by a parallel increase in apoptotic cells, as no significant differences in apoptosis were observed between HCT116 cells and the 14-fold resistant HCT-116 p21-/- cell line (figure
4). This suggests that modes of cell death other than apoptosis may operate in tumor cells following exposure to DOX, or, more generally, to DNA-damaging agents, a concept that is beginning to be proposed by a number of Authors [
39,
40]; of note, recent experimental evidence indicates p21 as one of the major determinants of terminal growth arrest induced by cytotoxic agents [
41‐
43]. Therefore, although issues related to terminal growth arrest and senescence have not been specifically addressed in the present study, the possibility that these phenomena might play a role linking cell death to the observed increases in p21 levels should not be disregarded.
The hypothesis that the cytotoxic response of the tumor cell lines tested in the present study may depend on p21 induction is further corroborated by data obtained following pre-treatment with the piperidine nitroxide TPL. The choice of this compound was dictated by previous findings indicating that TPL induces cell death in a number of tumor cell lines irrespective of their
p53 status [
15], and that it increases p21 levels in
p53-null cells [
16]. The results of the present study show that TPL affects the four colon cell lines to similar extents, thus confirming that its growth inhibitory effect is independent of p53 function. HCT-116 p21-/- cells are actually slightly less responsive than the other cell lines [even though the difference does not attain statistical significance), which suggests the possibility that the effects of TPL are due in part to its ability to increase p21 levels. Interestingly, the nitroxide also induces p21 expression even in p53-deficient cell lines; this observation suggests that TPL can activate p53-independent pathways for p21 induction, as already noted following exposure of HT29 and HCT116 E6 cells to DOX. Moreover, activation of such pathways by TPL appears to sensitize tumor cells to the action of DOX: in fact, synergistic potentiation of DOX cytotoxicity is achieved by TPL in those cell lines where p21 expression can be induced, but only additive effects between TPL and DOX are observed in HCT116 p21-/-, where p21 expression is constitutively absent.