We reported previously on the induction of apoptosis by CHL in HCT116 cells.36
In the present study, CHL affected the growth and survival of other colon cancer cell lines, causing an increase in the population of floating cells and a decrease in the attached cell yield. HCT116 cells (p53-wild type) were sensitive to CHL treatment whereas HT29 cells (p53-mutant) were more resistant. However, following treatment with 250 μM CHL, both cell lines exhibited a sub-G1
peak in the adherent cell population at 24 h, indicative of apoptosis. As a critical regulator of the cell cycle and apoptosis, p53 is often mutated in human cancers, although some p53 mutations can be gain-of-function.59
Thus, we became interested in the p53 status and how it influences sensitivity to CHL treatment.
In HCT116 cells, low doses of CHL induced a slight increase in the population of cells occupying G1
with loss of cells in S phase, whereas at higher CHL concentrations a striking S-phase arrest was detected. The differential response at low versus
high CHL dose is worthy of further investigation, but we elected to focus here on the S-phase arrest. Immunoblot analyses confirmed the loss of several checkpoint controls that regulate G1
/S transition, including p53, p21, and Cyclin D1. Cyclin D1 depletion also has been observed in CHL-treated MCF-7 breast cancer cells.60
Two other important cell cycle regulators, Rb and MDM2, were decreased at the protein level following CHL treatment. Despite the loss of Cyclin D1, hyperphosphorylated Rb (pRb) was detected even at the highest CHL concentrations. It is possible that pRb was generated by active Cyclin E/Cdk2 or Cyclin A/Cdk2 complexes.61
We were particularly interested in determining how CHL might affect the interplay between E2F family members and their associated binding partners. The levels of E2F2, E2F3, and E2F5 were not altered in whole cell lysates (data not presented), but E2F1 and E2F4 proteins were elevated markedly by CHL treatment. When bound to the DNA-binding partner DP-1, E2F1 becomes a transcriptional activator and E2F4 a transcriptional repressor.55,62
Interestingly, Cyclin A-Cdk2 interacts with an amino-terminal domain on E2F1 and inhibits E2F1/DP-1 transcriptional activity, but E2F4, which lacks this domain, associates indirectly with Cyclin A-Cdk2 through one of the pocket proteins, p130 or p107.62
We saw no changes in p130 levels after CHL treatment (data not presented), but p107 protein expression was decreased in a dose-dependent manner. This suggested a scenario in which the loss of p107 might limit the interactions of Cyclin A-Cdk2 with E2F4, freeing E2F4 to act as a transcriptional repressor. Support for this idea came from the mobility-shift assays, with the increased DNA binding activity of E2F4 in nuclear extracts from CHL-treated cells, and the loss of p107 and Cyclin A. Prior studies have shown that Rb is a binding partner for both E2F1 and E2F4, and that once cells enter S-phase there is a pool of free E2F1 and E2F4 in equal mixture.55
Mobility-shift assays provided evidence for increased E2F1 and Rb in nuclear extracts of CHL-treated cells, but the levels of binding typically were much weaker than observed for E2F4. This might be because E2F4 competed with E2F1 for a limited supply of nuclear DP-1, but further studies are needed to clarify this question.
The experiments to this point implicated E2F4 as a transcriptional repressor during CHL-induced S-phase arrest in HCT116 cells. Subsequent work using BrdU/PI covariate cell cycle analysis demonstrated that CHL inhibited DNA synthesis, and we turned our attention to RR. The promoter region of the mouse R2
gene has been reported to bind E2F4 as a negative regulator,8
and we considered the possibility that increased E2F4 levels in CHL-treated cells might transcriptionally downregulate R2
expression. Interestingly, both R1 and R2 subunits were decreased in a concentration-dependent fashion in HCT116 cells, R2 being particularly sensitive to CHL treatment.
When R2 is unavailable, the p53-inducible subunit p53R2 can associate with R1 to form an active RR enzyme.22
However, there was a marked loss of p53R2 expression in both HCT116 (p53+/+
) and HCT116 (p53−/−
) cells following CHL treatment, in addition to reduced R1 and R2 levels (). It is interesting to consider these findings in the context of prior work that used knockdown strategies to target either R2 or p53R2. Lin et al
observed that R2 levels critically determine the sensitivity of HCT116 (p53−/−
) cells to RR inhibitors, and that ectopic expression of p53R2 does not compensate for the decreased levels of R2 brought about by R2
-siRNA. Yoshida et al
knocked down either p53 or p53R2 in a panel of cell lines and demonstrated a reduction in the DNA repair response induced by short-chain fatty acids. Interestingly, HCT116 cells have a mutant form of p53R2; as a consequence, the DNA repair response is attenuated and p53
siRNAs had little or no effect on DNA repair activity.64
An important conclusion from the knockdown studies is that the level of R2 protein reflects the ability of cells to participate in DNA repair, and is a critical determinant of the sensitivity to DNA damaging agents.63,64
Given that CHL markedly inhibited R2 expression in HCT116 (p53+/+
) and HCT116 (p53−/−
) cells (), we conclude that CHL might be effective in sensitizing cancer cells to DNA-damaging agents, regardless of the p53 status.
It is noteworthy that, in addition to the transcriptional downregulation of RR subunits, CHL had a direct inhibitory effect on RR enzymatic activity. This was demonstrated by taking HCT116 cells that had not been treated with CHL, and using the cell extracts in the RR enzyme assay, where CHL produced dose-dependent inhibition. These findings resemble the reported effects of inhibitors such hydroxyurea, which quench the tyrosyl free radical in the active site of R2.58
In this context, it is interesting to consider briefly the evidence for CHL as a free radical-scavenging agent.
CHL has been shown to react with hydroxyl radical as well as deoxyribose peroxyl radical generated by pulse radiolysis in vitro.65
The stable 1,1-diphenyl-2-picrylhydrazyl radical also was scavenged by CHL. An electron paramagnetic resonance study provided evidence that CHL inhibited formation of 5,5-dimethyl-1-pyrroline-N
-oxide-hydroxyl radical and 2,2,6,6-tetramethyl-piperidine oxide radical, generated by γ-radiation or by photosensitization of methylene blue with visible light.66
CHL also inhibited oxidation of low-density lipoprotein, and scavenged tyrosyl radicals that were generated by myeloperoxidase in the presence of H2
Porphyrins in general are strong protein binders, and CHL itself interacts with human serum albumin with an affinity constant of 7.0 × 103
In the case of RR, CHL binding might displace rNDP substrates and/or allosteric modifiers from their corresponding binding sites by interacting with one or both subunits of the enzyme. Alternatively, CHL might cause conformational changes that no longer favor binding of substrates and/or allosteric effectors to R1. Further studies are needed to clarify the precise mechanism(s) by which CHL inhibits RR activity, and the extent to which this sensitizes cancer cells to DNA-damaging agents or anticancer therapies specifically targeting RR.20
In summary, we conducted a detailed investigation of cell cycle checkpoint controls that are altered during CHL-induced S-phase arrest in colon cancer cells. With loss of p53, p21, and other key regulators of the G1
/S transition there was increased expression of E2F1 and E2F4 transcription factors. The role of E2F4 as a reported transcriptional repressor of RR, coupled with the inhibition of DNA synthesis by CHL, led to studies of R1, R2, and p53R2. CHL reduced markedly the expression levels of all three subunits, with R2 being particularly sensitive, and there was loss of RR enzyme activity. We conclude that CHL might provide a useful new avenue for cancer treatment in the clinical setting, helping to sensitize cancer cells to the actions of anticancer agents.69 In vivo
studies to corroborate this possibility are now in progress.