John Patrick Alao

The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention

Cyclin D1 is an important regulator of cell cycle progression and can function as a transcriptionl co-regulator. The overexpression of cyclin D1 has been linked to the development and progression of cancer. Deregulated cyclin D1 degradation appears to be responsible for the increased levels of cyclin D1 in several cancers. Recent findings have identified novel mechanisms involved in the regulation of cyclin D1 stability. A number of therapeutic agents have been shown to induce cyclin D1 degradation. The therapeutic ablation of cyclin D1 may be useful for the prevention and treatment of cancer. In this review, current knowledge on the regulation of cyclin D1 degradation is discussed. Novel insights into cyclin D1 degradation are also discussed in the context of ablative therapy. A number of unresolved questions regarding the regulation of cellular cyclin D1 levels are also addressed.

Histone Deacetylase Inhibitor Trichostatin A Represses Estrogen Receptor α-Dependent Transcription and Promotes Proteasomal Degradation of Cyclin D1 in Human Breast Carcinoma Cell Lines

Purpose: Estrogen receptor α (ERα)-positive breast cancer cell lines are up to 10 times more sensitive than ERα-negative cell lines to the antiproliferative activity of the histone deacetylase inhibitor trichostatin A (TSA). The purpose of the study was to investigate the mechanisms underlying this differential response. Experimental Design and Results: In the ERα-positive MCF-7 cell line, TSA repressed ERα and cyclin D1 transcription and induced ubiquitin dependent proteasomal degradation of cyclin D1, leading primarily to G1-S-phase cell cycle arrest. By contrast, cyclin D1 degradation was enhanced but its transcription unaffected by TSA in the ERα-negative MDA-MB-231 cell line, which arrested in G2-M phase. Cyclin D1 degradation involved Skp2/p45, a regulatory component of the Skp1/Cullin/F-box complex; silencing SKP2 gene expression by RNA interference stabilized cyclin D1 and abrogated the cyclin D1 down-regulation response to TSA. Conclusions: Tamoxifen has been shown to inhibit ERα-mediated cyclin D1 transcription, and acquired resistance to tamoxifen is associated with a shift to ERα-independent cyclin D1 up-regulation. Taken together, our data show that TSA effectively induces cyclin D1 down-regulation through both ERα-dependent and ERα-independent mechanisms, providing an important new strategy for combating resistance to antiestrogens.

PLASMA PHARMACOKINETICS AND METABOLISM OF THE HISTONE DEACETYLASE INHIBITOR TRICHOSTATIN A AFTER INTRAPERITONEAL ADMINISTRATION TO MICE

Trichostatin A is a potent and specific histone deacetylase inhibitor with promising antitumor activity in preclinical models. Plasma pharmacokinetics of trichostatin A were studied following single-dose intraperitoneal administration of 80 mg/kg (high dose) or 0.5 mg/kg (low dose) to female BALB/c mice. Plasma trichostatin A concentrations were quantified by high performance liquid chromatography (HPLC)-UV assay (high dose) or by HPLC-multiple reaction monitoring assay (low dose). Trichostatin A was rapidly absorbed from the peritoneum and detectable in plasma within 2 min. Cmax of 40 μg/ml and 8 ng/ml occurred within 5 min, followed by rapid exponential decay in plasma trichostatin A concentration with t1/2 of 6.3 min and 9.6 min (high and low doses, respectively). Phase I metabolites at the high dose were identified by simultaneous UV and positive ion electrospray mass spectrometry. Trichostatin A underwent extensive metabolism: primary metabolic pathways were N-demethylation, reduction of the hydroxamic acid to the corresponding trichostatin A amide, and oxidative deamination to trichostatic acid. N-Monomethyl trichostatin A amide was the major plasma metabolite. No didemethylated compounds were identified. Trichostatic acid underwent further biotransformation: reduction and β-oxidation of the carboxylic acid, with or without N-demethylation, resulted in formation of dihydro trichostatic acid and dinor dihydro trichostatic acids. HPLC fractions corresponding to trichostatin A and N-demethylated trichostatin A exhibited histone deacetylase-inhibitory activity; no other fractions were biologically active. We conclude that trichostatin A is rapidly and extensively metabolized in vivo following intraperitoneal administration to mice, and N-demethylation does not compromise histone deacetylase-inhibitory activity.

The ATM and ATR inhibitors CGK733 and caffeine suppress cyclin D1 levels and inhibit cell proliferation

The ataxia telangiectasia mutated (ATM) and the ATM- related (ATR) kinases play a central role in facilitating the resistance of cancer cells to genotoxic treatment regimens. The components of the ATM and ATR regulated signaling pathways thus provide attractive pharmacological targets, since their inhibition enhances cellular sensitivity to chemo- and radiotherapy. Caffeine as well as more specific inhibitors of ATM (KU55933) or ATM and ATR (CGK733) have recently been shown to induce cell death in drug-induced senescent tumor cells. Addition of these agents to cancer cells previously rendered senescent by exposure to genotoxins suppressed the ATM mediated p21 expression required for the survival of these cells. The precise molecular pharmacology of these agents however, is not well characterized. Herein, we report that caffeine, CGK733, and to a lesser extent KU55933, inhibit the proliferation of otherwise untreated human cancer and non-transformed mouse fibroblast cell lines. Exposure of human cancer cell lines to caffeine and CGK733 was associated with a rapid decline in cyclin D1 protein levels and a reduction in the levels of both phosphorylated and total retinoblastoma protein (RB). Our studies suggest that observations based on the effects of these compounds on cell proliferation and survival must be interpreted with caution. The differential effects of caffeine/CGK733 and KU55933 on cyclin D1 protein levels suggest that these agents will exhibit dissimilar molecular pharmacological profiles.

Role of glycogen synthase kinase 3 beta (GSK3β) in mediating the cytotoxic effects of the histone deacetylase inhibitor trichostatin A (TSA) in MCF-7 breast cancer cells

Histone deacetylase inhibitors (HDACIs) have been shown to induce apoptotic and autophagic cell death in vitro and in vivo. The molecular mechanisms that underlie these cytotoxic effects are not yet clearly understood. Recently, HDACIs were shown to induce Akt dephosphorylation by disrupting HDAC-protein phosphatase 1 (PP1) complexes. This disruption results in the increased association of PP1 with Akt, resulting in the dephosphorylation and consequent inactivation of the kinase. Akt enhances cellular survival through the phosphorylation-dependent inhibition of several pro-apoptotic proteins. Akt is an important negative regulator of GSK3β, a kinase that has been shown to regulate apoptosis in response to various stimuli. In the present study, we investigated the role of GSK3β in mediating the cytotoxic effects in MCF-7 breast cancer cells treated with trichostatin A (TSA), a prototype HDACI. We show that TSA induces Akt dephosphorylation in a PP1-dependent manner, resulting in activation of GSK3β in MCF-7 cells. Similarly, knockdown of HDAC1 and-2 by small interfering RNA (siRNA) resulted in the dephosphorylation of Akt and GSK3β. Selective inhibition of GSK3β attenuated TSA induced cytotoxicity and resulted in enhanced proliferation following drug removal. Our findings identify GSK3β as an important mediator of TSA-induced cytotoxicity in MCF-7 breast cancer cells.

Preparation of 3-Substituted-1-Isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines as RET Kinase Inhibitors

A series of 3-substituted-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines have been designed, synthesized, and evaluated as RET protein kinase inhibitors. On the basis of docking results, a small library of pyrazolopyrimidine compounds with an extended hydrophobic side arm was synthesized. The most promising of the compounds (7a) displayed efficient inhibition in vitro and good selectivity when tested on a panel of kinases. Furthermore, 7a inhibited GDNF-induced RET phosphorylation of ERK1/2 in MCF-7 breast cancer cells at concentrations as low as 100 nM.

Design, Synthesis and Biological Evaluation of Chromone-based p38 MAP Kinase Inhibitors

3-(4-Fluorophenyl)-2-(4-pyridyl)chromone derivatives were synthesized and evaluated as p38 MAP kinase inhibitors. Introduction of an amino group in the 2-position of the pyridyl moiety gave p38α inhibitors with IC(50) in the low nanomolar range (e.g., IC(50) = 17 nm). The inhibitors showed excellent selectivity profiles when tested on a panel of 62 kinases, as well as efficient inhibition of p38 signaling in human breast cancer cells.

Flemingins G-O, cytotoxic and antioxidant constituents of the leaves of Flemingia grahamiana.

The known flemingins A-C (1-3) and nine new chalcones, named flemingins G-O (4-12), along with deoxyhomoflemingin (13) and emodin (14) were isolated from a leaf extract of Flemingia grahamiana. The isolated chalcones were found to have a geranyl substituent modified into a chromene ring possessing a residual chain, as shown by spectroscopic methods. The leaf extract showed an IC50 value of 5.9 μg/mL in a DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay. The chalcones flemingins A, B, C, G, and H were active in the DPPH radical scavenging assay (ED50 4.4-8.9 μM), while flemingins A and C showed cytotoxicity against MCF-7 human breast cancer cells (IC50 8.9 and 7.6 μM, respectively).

Antiplasmodial Quinones from Pentas longiflora and Pentas lanceolata

The dichloromethane/methanol (1:1) extracts of the roots of Pentas longiflora and Pentas lanceolata showed low micromolar (IC(50) = 0.9-3 µg/mL) IN VITRO antiplasmodial activity against chloroquine-resistant (W2) and chloroquine-sensitive (D6) strains of PLASMODIUM FALCIPARUM. Chromatographic separation of the extract of PENTAS LONGIFLORA led to the isolation of the pyranonaphthoquinones pentalongin (1) and psychorubrin (2) with IC(50) values below 1 µg/mL and the naphthalene derivative mollugin (3), which showed marginal activity. Similar treatment of Pentas lanceolata led to the isolation of eight anthraquinones ( 4-11, IC(50) = 5-31 µg/mL) of which one is new (5,6-dihydroxydamnacanthol, 11), while three--nordamnacanthal (7), lucidin-ω-methyl ether (9), and damnacanthol (10)--are reported here for the first time from the genus Pentas. The compounds were identified by NMR and mass spectroscopic techniques.

Anthraquinones of the roots of Pentas micrantha

Pentas micrantha is used in the East African indigenous medicine to treat malaria. In the first investigation of this plant, the crude methanol root extract showed moderate antiplasmodial activity against the W2- (3.37 μg/mL) and D6-strains (4.00 μg/mL) of Plasmodium falciparum and low cytotoxicity (>450 μg/mL, MCF-7 cell line). Chromatographic separation of the extract yielded nine anthraquinones, of which 5,6-dihydroxylucidin-11-O-methyl ether is new. Isolation of a munjistin derivative from the genus Pentas is reported here for the first time. The isolated constituents were identified by NMR and mass spectrometric techniques and showed low antiplasmodial activities.