Since in many patients the AML blasts have wild-type p53 (34
), any consideration of the use of vitamin D derivatives for treatment of these patients requires a demonstration that such blasts can respond to these compounds. Thus, it is important, as shown here, that AML cells expressing wild-type p53 can be terminally differentiated by exposure to 1,25D, as previously shown for cells with mutated or deleted p53 (14
). In the presence of 1,25D, MOLM-13 and OCI-AML3 cells expressed the classical differentiation markers similar to that of HL60 cells (14
), indicating that p53 status does not interfere with the ability of 1,25D to activate vitamin D signaling and induce differentiation. Our model cell system utilized AML cells with expression of normal endogenous levels of p53, which is selectively activated by nutlin-3a. In contrast, Chylicki et al., (37
) used a model in which a temperature-inducible form of p53 was transfected into U937 cells. Since p53 is normally expressed at very low levels, their system may not accurately reflect the dynamic changes and functions of cellular p53.
MDM2 antagonists, the nutlins, are a novel class of small molecules that selectively activate the p53 pathway to induce apoptosis and may offer a new treatment modality for patients with AML (9
). Here, we have studied the effect of combining the pro-apoptotic MDM2 antagonist, nutlin-3a, with the differentiating agent 1,25D in AML cells expressing wild-type p53, MOLM-13 and OCI-AML3. Addition of 1,25D to nutlin resulted in accelerated apoptotic response in both cell lines (, ). We have identified possible contributors to this enhanced apoptotic effect: the downregulation of anti-apoptotic BCL-2, MDMX, hKSR2, p-ERK1/2, and the enhanced upregulation of PIG-6 by nutlin and 1,25D combination. BCL-2 is overexpressed in OCI-AML3 cells; siRNA-mediated knockdown of BCL-2 was sufficient to induce apoptosis in the absence of 1,25D or nutlin-3a (). This led to enhanced apoptosis induced by nutlin alone or nutlin in combination with 1,25D (). BCL-2 proteins localize on mitochondria and smooth endoplasmic reticulum, dimerize with BCL-W, BCL-XL, or MCL-1 to from multi-domain complexes with pro-apoptotic BAX and Bak (38
). Therefore, therapeutic intervention which could lower the expression of BCL-2 would be beneficial for the patient by enhancing the action of cell death-inducing agents.
In the presence of 1,25D, the protein levels of p53 regulators MDM2 and MDMX dropped significantly (). MDMX downregulation could also contribute to the overall apoptotic response. Both MDM2 and MDMX are not known as 1,25D targets but there is emerging evidence that mitogenic signaling pathways regulate cellular localization of p53. Studies by Kojima et al., (39
) demonstrated that a MEK inhibitor and nutlin-3a sensitize AML cells to apoptosis due to transcriptional activation of p53 target genes in the nucleus.
Another downstream target of p53 is pro-apoptotic PIG-6, also referred to as proline dehydrogenase (PRODH) and proline oxidase-2 (POX2), that is localized on the inner mitochondrial membrane. Polyak et al
) used serial analysis of gene expression (SAGE) to demonstrate that PIG-6 is a p53-induced gene. At low levels, the enzyme couples the oxidation of NADPH to mitochondrial electron transport, and thus provides cells with ATP. At high levels, PIG-6 activates oxidative apoptosis by generating reactive oxygen species (ROS) such as superoxides. These oxygen radicals are known to damage cellular components such as DNA, protein, lipids. Other groups have demonstrated that overexpression of PIG-6 in a variety of cancer cell types induces apoptosis in a p53-dependent manner (41
). Induction of PIG-6 has been shown to reduce phosphorylated levels of MEK1/2 and ERK1/2 during p53-induced oxidative apoptosis in solid tumor cell lines (33
). Therefore, reduced levels of phospho MEK1/2 and ERK1/2 in our experiments may be a consequence of PIG-6 induction () and activation of p53, but this remains to be further investigated.
Our results indicate that various cellular events may contribute to apoptosis in 1,25D and nutlin-3a- treated OCI-AML3 cells. It is well documented that the MAPK family proteins are activated during 1,25D-induced differentiation of AML cells with mutant or non-functional p53 (14
). 1,25D also increases survival in HL-60 cells and other p53-null cells by upregulation of p21, p27, p35, p-ERK1/2 and hKSR2 (16
). In addition, the MEK/ERK pathway is known to promote growth and to prevent apoptosis in hematological malignancies such as AML (44
). Both MOLM-13 and OCI-AML3 cell lines constitutively express phosphorylated ERK1/2 due to the aberrant expression of mutant FLT3 (45
). Western blot analysis depicted the reduction of p-MEK and p-ERK by nutlin-3a alone whereas 1,25D had no effect on the phosphorylated levels of these proteins under the conditions studied here (data not shown). Upon combination with 1,25D, the level of p-ERK1/2 remained reduced when compared with basal level ( and ). One can speculate that sustained repression of p-ERK1/2 in the presence of 1,25D could be due to induced levels of PIG-6. Human KSR2 was also further reduced by 1,25D in nutlin-3a-treated cells during apoptosis. Since hKSR2 appears to function as a survival protein, one could expect a p53 activator such as nutlin-3a to inhibit its expression. Wang et al., (30
) demonstrated that siRNA knockdown of hKSR2 sensitized arabinocytosine-treated HL60 cells to undergo apoptosis.
Our data suggest that several factors contribute to the sensitization of OCI-AML3 cells to apoptosis when exposed to 1,25D and nutlin-3a. These are BCL-2, MDMX, phosphorylated ERK1/2, hKSR2 and PIG-6. One possibility is that 1,25D recruits its co-activators steroid receptor coactivator-1 (SRC-1) and vitamin D receptor interacting protein (DRIP) 205 to response elements in the p53 promoter thereby enabling p53 to be more effective at inducing cell death.
The use of 1,25D for treatment of AML is limited due to hypercalcemia, hence deltanoids devoid of this undesirable side effect are being developed. Preclinical work on cultured cells and in animals demonstrated that differentiation induced by 1,25D and its analogs can be markedly increased by combination with plant-derived antioxidants, such as carnosic acid extracted from Rosemarinus officinalis (46
). Ongoing studies also indicate synergistic or additive antiproliferative effect of 1,25D and chemotherapeutic agents in various solid tumors (48
). Our study suggests that vitamin D may enhance the activity of a novel class of antitumor agents, the MDM2 antagonists.