Oluwakemi Obajimi

Cooper University Hospital

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Peter W. Melera

University of Maryland, Baltimore

Iain Glen

University of the Highlands and Islands

Brian M. Ross

NOSM University

Kenneth Black

Scottish Association For Marine Science

Judith C. Keen

American Society for Radiation Oncology

Rose M. Boyle

Glasgow Victoria Infirmary

Peter Pryzbylkowski

University of Pennsylvania

Donald J. MacDonald

Glasgow Victoria Infirmary

Cooperative Institutions

Millennium Institute

NHS Highland

Glasgow Victoria Infirmary

Rutgers, The State University of New Jersey

University of the Highlands and Islands

Universität Hamburg

Raigmore Hospital

University College London

NOSM University

Lakehead University

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Inhibition of de novo purine synthesis in human prostate cells results in ATP depletion, AMPK activation and induces senescence

The Prostate (2009)

Oluwakemi Obajimi Judith C. Keen Peter W. Melera

BACKGROUND 4-[2-(2-Amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-l-glutamic acid (AG2034), is a classical antifolate shown to be an excellent inhibitor of glycinamide ribonucleotide formyltransferase (GARFT), ultimately inhibiting de novo purine synthesis. We examined some metabolic effects of this drug in prostate cancer cells, LNCaP, versus non-tumorigenic prostatic epithelial cells, RWPE-1. METHODS AND RESULTS Cells were cultured in medium containing 10 nM 5-methyl-tetrahydrofolate supplemented with/without 1.7 µM hypoxanthine/1.5 µM thymidine. Cytotoxicity of AG2034 was determined by clonogenic assays. Total ATP was quantified by reverse-phase HPLC and [14C]-glycine incorporation and [3H]-hypoxanthine conversion into ATP by liquid scintillation counting. Protein expression levels were determined by Western blotting, cell cycle analysis by propidium iodide staining and cell-senescence by β-galactosidase staining. AG2034 inhibited LNCaP cell proliferation causing death in the absence of hypoxanthine and cytostasis in its presence. However, RWPE-1 cells were resistant to AG2034 when hypoxanthine was present. AG2034 elevates AMP/ATP ratios but is unable to activate AMPK in RWPE-1 when hypoxanthine is present. Drug exposure increased expression levels of p53, p21, p27, and p16 in both cell lines and increased senescence-associated-β-gal staining in LNCaP with/without hypoxanthine, but primarily in its absence in RWPE-1. CONCLUSIONS LNCaP cells primarily depend upon de novo while RWPE-1 cells largely favor salvage synthesis for maintenance of their ATP pools. With AG2034 treatment, ATP synthesis via hypoxanthine salvage is insufficient to support growth of LNCaP but enough to restore ATP levels and support RWPE-1 growth. The anti-proliferative effect of AG2034 involves increasing phosphorylation of AMPK. These results indicate that AG2034 activates p53 and AMPK mediating the induction of signaling pathways leading to senescence. Prostate 69:1206–1221, 2009. © 2009 Wiley-Liss, Inc.

Hypoxanthine

Cytostasis

Clonogenic assay

DU145

Propidium iodide

Purine metabolism

10.1002/pros.20971

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Citations (11)

Trichostatin A and 5 Aza-2′ deoxycytidine decrease estrogen receptor mRNA stability in ER positive MCF7 cells through modulation of HuR

Breast Cancer Research and Treatment (2007)

Peter Pryzbylkowski Oluwakemi Obajimi Judith C. Keen

Trichostatin A

Estrogen receptor alpha

10.1007/s10549-007-9751-0

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Citations (73)

Differential effects of eicosapentaenoic and docosahexaenoic acids upon oxidant-stimulated release and uptake of arachidonic acid in human lymphoma U937 cells

Pharmacological Research (2005)

Oluwakemi Obajimi Kenneth Black Donald J. MacDonald Rose M. Boyle Iain Glen

CYP2C8

10.1016/j.phrs.2005.02.024

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Citations (23)

Abstract 77: Suppression of ERK1/2 with siRNA restores drug sensitivity in DU145 cells selected for resistance to AG2034

Cancer Research (2010)

Oluwakemi Obajimi Peter W. Melera

Abstract We examined the involvement of the extracellular signal regulated kinase (ERK) cascade (Ras/Raf/MEK/ERK) in the promotion of tumor cell growth, survival and the maintenance of ATP pools in DU145 prostate cancer cells selected for resistance to 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4] thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034). The Ras/Raf/MEK/ERK cascade is a subfamily of the MAPK signal transduction pathway and it plays an important role in gene expressions, growth and differentiation of many cell types. The components of this pathway are often altered in tumor cells and conflicting evidence exists regarding its contribution to the development of resistance to several chemotherapeutic drugs. DU145 cells were selected over time for resistance to AG2034 by gradually decreasing hypoxanthine concentrations from 1.7 µM to zero in the continuous presence of 10 nM 5-methyl tetrahydrofolate and 50 nM AG2034. Since the inhibition of glycinamide ribonucleotide formyl-transferase blocks the incorporation of glycine into the purine ring, total cellular ATP was quantified by reverse-phase HPLC and [14C]-Glycine incorporation into ATP was determined by liquid scintillation counting of HPLC-isolated ATP fractions. Total and phosphorylated proteins were determined by western blotting. Quantitative RT-PCR was utilized to characterize mRNA expression levels before and after transient transfection studies with siRNA. The continuous treatment of cells with AG2034 is cytotoxic to parent DU145 cells and inhibits proliferation. However, in the resistance clones, drug presence did not prevent cell growth but rather, markedly reduces the growth rate in comparison to drug-naïve parent cells. While the treatment of parent cells with AG2034 for 24 h depletes total ATP levels, there was no significant difference in total ATP levels between parent cells and resistant clones. Although AG2034 inhibits [14C]-glycine incorporation into ATP in parent cells, this effect is abrogated by drug resistance. Acquired resistance increases ERK expression levels and phosphorylation status. It also increases upstream activators, NRas, BRaf and MEK. In addition, decreasing the expression of ERK by siRNA reduces steady state levels of total and phosphorylated ERK and results in a marked depletion of total ATP levels in resistant clones. In the parent cells however, ERK inhibition resulted in increased ERK phosphorylation but had no effect on total ATP pools. The inhibition of ERK re-sensitizes drug-resistant DU145 cells to AG2034 concomitant with a depletion of cellular ATP pools. These results demonstrate that DU145 cells selected for resistance to AG2034 require the sustenance of ERK1/2 phosphorylation for the maintenance of their ATP pools and suggest that the intrinsic activation of ERK signaling supports growth and survival in these AG2034-resistant clones. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 77.

DU145

Hypoxanthine

10.1158/1538-7445.am10-77

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The effects of fatty acids and antioxidants on U937 monocytes used as a model for the lipid disturbance in schizophrenia

Oluwakemi Obajimi

Schizophrenia is a devastating mental disease affecting approximately 1% of all populations. Recent studies have suggested that deficient uptake of free fatty acids or excessive breakdown of cell-membrane phospholipids, directly or indirectly triggered by lipid peroxidative damage, may be associated with schizophrenia and possibly other neurodegenerative disorders. This study provides more information on the influence of the presence of different fatty acids (FAs): stearic acid (SA), oleic acid (OA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) and dietary antioxidants (ascorbic acid, atocopherol, f3-carotene and astaxanthin) as components of the culture-medium on the human monocytic cell line (U937) by the measurements of cell-viability, productions of by-products of lipid peroxidation (lipid hydroperoxides and volatile hydrocarbons), AA release/uptake and cPLA2 expression upon induced-oxidative stress with the radical mediating system oft-butyl hydroperoxide (t-BHP)/Fe2+. Of all FAs used, the PUFAs (AA, EPA and DHA) at 40 µM concentrations reduced cell membrane integrity significantly whereas SA and OA had no effect after 72 h of incubation, suggesting the cytotoxicity of the PUF As above certain concentrations. This study shows that U937 cells accumulate lipid hydroperoxides, generate ethane, butane and pentane, release and incorporate AA at a greater rate in response to induced-oxidative stress by a mechanism probably involving cPLA2. In addition, AA is incorporated into cellular phospholipids at a greater rate post-oxidation. With oxidative-stress, the presence of EPA but not DHA in cellular phospholipids significantly increases the accumulation of AA in the extracellular fluid. Studies revealed that under oxidative-stress α-tocopherol is a potent antioxidant markedly inhibiting AA-release but not its uptake. Whilst ascorbic acid appeared to promote AA release, β-carotene and astaxanthin conferred no protective effect. Collectively, this study demonstrates the potency of α-tocopherol, ascorbic acid, β-carotene and astaxanthin against oxidant-induced peroxidative damage, AA release/uptake and ultimately total loss of cell-viability. In EPA-pretreated cells, AA-release is enhanced with/without oxidant activation suggesting its action by stimulating the availability of AA possibly for prostaglandin production.

Stearic acid

Unsaturated fatty acid

10.21954/ou.ro.0000f57a

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Antioxidant modulation of oxidant-stimulated uptake and release of arachidonic acid in eicosapentaenoic acid-supplemented human lymphoma U937 cells

Prostaglandins Leukotrienes and Essential Fatty Acids (2007)

Oluwakemi Obajimi Kenneth Black Iain Glen Brian M. Ross

U937 cell

10.1016/j.plefa.2006.11.001

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Citations (12)

ERK Suppression in DU145 Cells Selected for AG2034 Resistance is Associated with Decreased ATP, GARFT Inactivation and Increased ERK Signaling

The Open Drug Resistance Journal (2011)

Oluwakemi Obajimi Peter W. Melera

Background: Previously, we showed that AG2034, an inhibitor of glycinamide ribonucleotide formyltransferase (GARFT) in the pathway for de novo purine synthesis, is cytotoxic to different prostate cancer cell lines in the absence of inosine and hypoxanthine in the culture media. Therefore, we examined DU145 androgen-independent prostate cancer cells selected for drug resistance over a prolonged period of time, in the absence of media components (inosine and hypoxanthine) required for salvage purine synthesis and in the continuous presence of AG2034.

DU145

Hypoxanthine

Inosine

Ribonucleotide reductase

Purine metabolism

10.2174/1876521101101010001

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ATP depletion by AG2034 mediates cell death or cytostasis in a hypoxanthine‐dependent manner in human prostate cancer cells

The FASEB Journal (2007)

Oluwakemi Obajimi Peter W. Melera

AG2034 is a classical antifolate shown to be an excellent inhibitor of glycinamide ribonucleotide formyltransferase, ultimately inhibiting de novo purine synthesis. Previous studies investigating the efficacy of AG2034 against various cancer types concluded that AG2034 is cytotoxic to p53-null cells and caused cytostasis in cells with wild-type p53 due to the arrest in G1 phase of the cell cycle. In this study, we examine the effect of inhibiting de novo purine synthesis with AG2034 on proliferation and cell cycle distribution in two prostate cancer cell lines, when maintained in culture medium containing physiological concentrations of reduced folate supplemented with or without hypoxanthine. AG2034 was cytotoxic to both DU145 (with mutant p53) and PC-3 (p-53 negative) without exhibiting G1 arrest, when maintained in a hypoxanthine-deficient medium. Although total ATP was depleted to about the same level with drug treatment, the presence of hypoxanthine promoted cytostasis while its absence resulted in cell kill in both cell lines studied. Taken together, our results reveal that the prolonged effect of AG2034 at inhibiting the production of ATP, cell proliferation and cell cycle distribution is different depending on the cancer cell type and most importantly, independent of the residual level of total ATP quantified in these cells. Source: The Greenbaum Cancer Centre, Univ. of MD, Baltimore, MD 21201 and CINJ @ Cooper, Camden, NJ 08103.

Cytostasis

Hypoxanthine

DU145

10.1096/fasebj.21.6.a1041-a

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Lapatinib as a Chemotherapeutic Drug

Recent Patents on Anti-Cancer Drug Discovery (2009)

Oluwakemi Obajimi

Human epidermal growth factor receptor (HER) signaling is frequently associated with the development and progression of several types of cancers. Both the MAPK and the PI3K/Akt pathways have been implicated as effectors of HER signaling by promoting anti-apoptotic and pro-proliferative effects in cancer cells. As a result, many anti-HER drugs have been developed and patented for use in cancer therapy. One such drug that was recently approved for clinical trials is lapatinib (Tykerb, GW572016). Lapatinib is a small molecule inhibitor that is active at the ATP binding site of the tyrosine kinase involved in HER signaling. Importantly, this drug has dual specificity acting at the ATP binding sites of both HER-2 and HER-1 (EGFR). This review therefore summarizes the current knowledge based on pre-clinical and clinical evidence of the therapeutic effects of lapatinib against cancer and the promising strategy of combination therapy with the possibility of circumventing the problems of drug resistance commonly faced by chemotherapeutic drugs.

Lapatinib

10.2174/157489209789206896

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Citations (1)

The depletion of cellular ATP by AG2034 mediates cell death or cytostasis in a hypoxanthine-dependent manner in human prostate cancer cells

Cancer Chemotherapy and Pharmacology (2007)

Oluwakemi Obajimi Peter W. Melera

Hypoxanthine

DU145

Cytostasis

Clonogenic assay

10.1007/s00280-007-0593-6

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Citations (12)