The effect of the cyclin-dependent kinase inhibitor flavopiridol on anaplastic large cell lymphoma cells and relationship with NPM-ALK kinase expression and activity.

BACKGROUND: The loss of cell cycle regulation due to abnormal function of cyclin-dependent kinases (cdk) occurs in tumors and leads to genetic instability of chemotherapy-resistant cells. In this study, we investigated the effect of the cdk inhibitor flavopiridol in anaplastic large cell lymphomas, in which unrestrained proliferation depends on NPM-ALK tyrosine kinase activity. DESIGN AND METHODS: Effects of flavopiridol were examined in ALK-positive and -negative anaplastic large cell lymphoma cells by means of immunoblotting and immunofluorescence analyses to assess cdk expression and activity, quantitative real time reverse transcriptase polymerase chain reaction to measure drug-induced changes in transcription, and FACS analyses to monitor changes in proliferation and survival. RESULTS: Treatment with flavopiridol resulted in growth inhibition of anaplastic large cell lymphoma cells, along with accumulation of subG(1) cells and disappearance of S phase without cell cycle arrest. Consistent with flavopiridol activity, phosphorylation at cdk2, cdk4, cdk9 sites on RB and RNA polymerase II was inhibited. This correlated with induction of cell death through rapid mitochondrial damage, inhibition of DNA synthesis, and down-regulation of anti-apoptotic proteins and transcripts. Notably, flavopiridol was less active in ALK-positive cells, as apoptosis was observed at higher concentrations and later time points, and resistance to treatment was observed in cells maintaining NPM-ALK signaling. NPM-ALK inhibition affected proliferation but not survival of anaplastic large cell lym-phoma cells, whereas it resulted in a dramatic increase in apoptosis when combined with flavopiridol. CONCLUSIONS: This work provides the first demonstration that targeting cdk is effective against anaplastic large cell lymphoma cells, and proves the critical role of NPM-ALK in the regulation of responsiveness of tumor cells with cdk dysregulation.

Identification of ellagic acid as potent inhibitor of protein kinase CK2: a successful example of a virtual screening application.

Casein kinase 2 (CK2) is a ubiquitous, essential, and highly pleiotropic protein kinase whose abnormally high constitutive activity is suspected to underlie its pathogenic potential in neoplasia and other diseases. Using a virtual screening approach, we have identified the ellagic acid, a naturally occurring tannic acid derivative, as a novel potent CK2 inhibitor. At present, ellagic acid represents the most potent known CK2 inhibitor (K(i) = 20 nM).

Inducible hsp70 in the regulation of cancer cell survival: analysis of chaperone induction, expression and activity.

Understanding the mechanisms that control stress is central to realize how cells respond to environmental and physiological insults. All the more important is to reveal how tumour cells withstand their harsher growth conditions and cope with drug-induced apoptosis, since resistance to chemotherapy is the foremost complication when curing cancer. Intensive research on tumour biology over the past number of years has provided significant insights into the molecular events that occur during oncogenesis, and resistance to anti-cancer drugs has been shown to often rely on stress response and expression of inducible heat shock proteins (HSPs). However, with respect to the mechanisms guarding cancer cells against proteotoxic stresses and the modulatory effects that allow their survival, much remains to be defined. Heat shock proteins are molecules responsible for folding newly synthesized polypeptides under physiological conditions and misfolded proteins under stress, but their role in maintaining the transformed phenotype often goes beyond their conventional chaperone activity. Expression of inducible HSPs is known to correlate with limited sensitivity to apoptosis induced by diverse cytotoxic agents and dismal prognosis of several tumour types, however whether cancer cells survive because of the constitutive expression of heat shock proteins or the ability to induce them when adapting to the hostile microenvironment remains to be elucidated. Clear is that tumours appear nowadays more "addicted" to heat shock proteins than previously envisaged, and targeting HSPs represents a powerful approach and a future challenge for sensitizing tumours to therapy. This review will focus on the anti-apoptotic role of heat shock 70kDa protein (Hsp70), and how regulatory factors that control inducible Hsp70 synthesis, expression and activity may be relevant for response to stress and survival of cancer cells.

Urolithin as a converging scaffold linking ellagic acid and coumarin analogues: design of potent protein kinase CK2 inhibitors.

Casein kinase 2 (CK2) is a ubiquitous, essential, and highly pleiotropic protein kinase; its abnormally high constitutive activity is suspected to underlie its pathogenic potential in neoplasia and other relevant diseases. Previously, using different in silico screening approaches, two potent and selective CK2 inhibitors were identified by our group: ellagic acid, a naturally occurring tannic acid derivative (K(i)=20 nM) and 3,8-dibromo-7-hydroxy-4-methylchromen-2-one (DBC, K(i)=60 nM). Comparing the crystallographic binding modes of both ellagic acid and DBC, an X-ray structure-driven merging approach was taken to design novel CK2 inhibitors with improved target affinity. A urolithin moiety is proposed as a possible bridging scaffold between the two known CK2 inhibitors, ellagic acid and DBC. Optimization of urolithin A as the bridging moiety led to the identification of 4-bromo-3,8-dihydroxy-benzo[c]chromen-6-one as a novel, potent and selective CK2 inhibitor, which shows a K(i) value of 7 nM against the protein kinase, representing a significant improvement in affinity for the target compared with the two parent fragments.