Cyclin-Dependent Kinase Inhibitors Sensitize Tumor Cells to Nutlin-Induced Apoptosis: a Potent Drug Combination

Abstract

Current chemotherapy focuses on the use of genotoxic drugs that may induce general DNA damage in cancer cells but also high levels of toxicity in normal tissues. Nongenotoxic activation of p53 by targeting specific molecular pathways therefore provides an attractive therapeutic strategy in cancers with wild-type p53. Here, we explored the antitumor potential of cyclin-dependent kinase (CDK) inhibitors in combination with a small molecule inhibitor of p53-murine double minute 2 (MDM2) interaction. We show that low doses of CDK inhibitors roscovitine and DRB synergize with the MDM2 antagonist nutlin-3a in the induction of p53 activity and promote p53-dependent apoptosis in a dose-and timedependent manner. Statistical measurement of the combination effects shows that the drug combination is additive on the reduction of cell viability and synergistic on inducing apoptosis, a critical end point of cytotoxic drugs. The degree of apoptosis observed 24 to 48 h after drug treatment correlated with the accumulation of p53 protein and concomitant induction of proapoptotic proteins Puma and PIG3. The antiproliferative and cytotoxic effects of this drug combination are validated in a range of tumor-derived cells including melanoma, colon carcinoma, breast adenocarcinoma, and hepatocarcinoma cells. Furthermore, this drug combination does not induce phosphorylation of Ser 15 on p53 and does not induce genotoxic stress in the cell. Given that many cytotoxic drugs rely on their ability to induce apoptosis via DNA damage -mediated activation of p53, the data presented here may provide a new therapeutic approach for the use of CDK inhibitors and MDM2 antagonists in combinatorial drug therapy.

Nutlin-3 Affects Expression and Function of Retinoblastoma Protein

“…It is possible that MDM2 expression or MDM2 activity are abnormal in LNCaP cells via other means, such as single nucleotide polymorphism of MDM2 (16), or factors affecting Rb kinase activities such as cycling D1 (29), Cdks (30), and p21 (31), all of which have impacts on the outcome of nutlin-3 treatment. In addition, other factors such as MDM4 (17,18), p73 (19) may also contribute to the sensitivity of nutlin-3-induced apoptosis in the absence of amplification of MDM2.…”

p53-based Cancer Therapy

“…Indeed, drug combinations involving genotoxic agents (e.g., doxorubicin) have been explored and are found to be synergistic in inducing p53 accumulation and activation of apoptosis. A recent report (Cheok et al 2007) on the combination of CDK inhibitors (Roscovitine and DRB) and nutlin-3 showed a clear synergism in the activation of p53 and apoptosis in p53 wild-type tumor cells. The combination retains the nongenotoxic nature characteristic of the individual compounds.…”

Wild type p53 reactivation: From lab bench to clinic

“…Therefore it could be a promising strategy to combine wild type reactivating compounds with those restoring mutant p53 function, such as PRIMA-1 MET /Apr246. Search for the rational combinations of drugs which can produce synergy when combined with Mdm2 inhibitors, revealed that low doses of CDK inhibitors promote apoptosis induction when combined with nutlin [147]. siRNA screen against known human kinases pinpointed to several pathways, including the MAP kinase pathway and the sphingosine kinase pathway whose inhibition may synergize with nutlin [148].…”