Simple and sensitive method for quantification of fludarabine triphosphate intracellular concentration in leukemic cells using isocratic liquid chromatography

Yamauchi, Takahiro; Ueda, Takanori

doi:10.1016/j.jchromb.2003.10.011

Cited by 15 publications

(19 citation statements)

References 15 publications

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“…15. 1 ϫ 10 7 cells were harvested after treatment and washed with PBS, and acid extraction of cell pellet was performed with 220 l of 0.3 M cold perchloric acid.…”

Section: Methodsmentioning

confidence: 99%

Targeting Glucose Consumption and Autophagy in Myeloma with the Novel Nucleoside Analogue 8-Aminoadenosine

Shanmugam

McBrayer

Qian

et al. 2009

Journal of Biological Chemistry

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Multiple myeloma, an incurable plasma cell malignancy, is characterized by altered cellular metabolism and resistance to apoptosis. Recent connections between glucose metabolism and resistance to apoptosis provide a compelling rationale for targeting metabolic changes in cancer. In this study, we have examined the ability of the purine analogue 8-aminoadenosine to acutely reduce glucose consumption by regulating localization and expression of key glucose transporters. Myeloma cells counteracted the metabolic stress by activating autophagy. Co-treatment with inhibitors of autophagy results in marked enhancement of cell death. Glucose consumption by drug-resistant myeloma cells was unaffected by 8-aminoadenosine, and accordingly, no activation of autophagy was observed. However, these cells can be sensitized to 8-aminoadenosine under glucose-limiting conditions. The prosurvival autophagic response of myeloma to nutrient deprivation or to nucleoside analogue treatment has not been described previously. This study establishes the potential of metabolic targeting as a broader means to kill and sensitize myeloma and identifies a compound that can achieve this goal. Multiple myeloma (MM)2 is a plasma cell malignancy with a yearly incidence of 14,000 in the United States and accounts for 10% of deaths from hematological malignancies (1). Multiple myeloma remains incurable with a median survival of 3-5 years. Current therapeutic options extend longevity, but patients eventually succumb to the disease due to the development of drug resistance (2).Nucleoside analogues are antimetabolites that play a pivotal role in the treatment of a spectrum of hematological malignancies. Our laboratory has extensively characterized the novel purine nucleoside analogues 8-chloroadenosine and congener 8-aminoadenosine (8-NH 2 -Ado) (3-5), which are highly effective in tissue culture models of multiple myeloma as well as a spectrum of other hematological and solid cancers. 8-Chloroadenosine is currently in phase I clinical trial. The general mechanism of action of these antimetabolites involves RNA and/or DNA termination, ATP depletion, and subsequent induction of apoptosis (6), but a comprehensive analysis of their cellular effects has not been reported.In this study, we describe a novel feature in the mechanism of action of purine nucleoside analogue 8-NH 2 -Ado. We find that 8-NH 2 -Ado inhibits glucose consumption that is associated with an activation of autophagy. This ability to reduce glucose consumption is particularly important, given the dependence of many tumor cells on aerobic glycolysis and the ensuing increase in glucose consumption to meet energetic and biosynthetic demands. Normal cells typically rely on oxygen to metabolize glucose via the glycolytic and oxidative phosphorylation pathways to generate ATP. In contrast, many tumor cells demonstrate enhanced glycolysis and lactate production, utilizing the less efficient mode of ATP production even in the presence of oxygen (7). This phenomenon, termed aerobic glycolysis, was ...

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“…15. 1 ϫ 10 7 cells were harvested after treatment and washed with PBS, and acid extraction of cell pellet was performed with 220 l of 0.3 M cold perchloric acid.…”

Section: Methodsmentioning

confidence: 99%

Targeting Glucose Consumption and Autophagy in Myeloma with the Novel Nucleoside Analogue 8-Aminoadenosine

Shanmugam

McBrayer

Qian

et al. 2009

Journal of Biological Chemistry

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“…The HPLC apparatus consisted of a pump (CCPM-II; Tosoh Corp., Tokyo, Japan), an autosampler (AS-8020; Tosoh Corp.), an in-line degasser (SD-8022; Tosoh Corp.), and a variablewavelength detector (UV-8020; Tosoh Corp.). LC-8020 software (Tosoh Corp.) installed on a personal computer was used to control the system and analyze the data (16). Samples were eluted isocratically with 0.06 M Na 2 HPO 4 (pH 6.9) containing 20% acetonitrile, at a constant flow rate of 0.7 ml/min and at ambient temperature.…”

Section: Methodsmentioning

confidence: 99%

A new high-performance liquid chromatography method determines low production of 9-β-D-arabinofuranosylguanine triphosphate, an active metabolite of nelarabine, in adult T-cell leukemia cells

Yamauchi¹

2009

Oncol Rep

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Abstract. The 9-ß-D-arabinofuranosylguanine (ara-G), an active compound of nelarabine, demonstrates potent cytotoxicity specifically on T-cell malignancies. In cells, ara-G is phosphorylated to ara-G triphosphate (ara-GTP), which is subsequently incorporated into DNA, thereby inhibiting DNA synthesis. Because ara-GTP is crucial to ara-G's cytotoxicity, the determination of ara-GTP production in cancer cells is informative for optimizing nelarabine administration. Here, we developed a new, sensitive isocraticelution HPLC method for quantifying ara-GTP. Samples were eluted isocratically by using phosphate buffer at a constant flow rate. Ara-GTP was clearly separated from other nucleotides by using an anion-exchange column and it was quantitated by its peak area at 254 nm. The standard curve was linear with low variability and a sensitive detection limit (10 pmol). Furthermore, due to ara-G's specificity to T-cells we hypothesized that nelarabine might be effective against adult T-cell leukemia (ATL). The ara-GTP production was compared between T-lymphoblastic leukemia CCRF-CEM and ATL cell lines in vitro. When CEM cells were incubated with ara-G, the ara-GTP production increased in a concentration-and time-dependent manner. In contrast, 5 ATL cell lines accumulated lower ara-GTP in the same condition. While ara-G inhibited the growth of CEM cells with a 50% growth inhibition concentration of 2 μM, the inhibitory-concentration values were >1 mM in 8 of the 12 ATL cell lines. This ineffectiveness appeared to correspond with the low ara-GTP production. The present study is the first to evaluate the potential of ara-G against ATL cells; our results suggest that nelarabine would not be effective against ATL.

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“…The intracellular accumulation of F-ara-ATP in leukemia cells was quantified in vitro using an HPLC system and a previously described method [25]. After drug treatment with the indicated dose of F-araA for 2 h at 37°C, the cells were washed twice with ice-cold PBS and harvested by centrifugation.…”

Section: Determination Of the Intracellular F-ara-atp Level In Leukemmentioning

confidence: 99%

“…We compared the intracellular concentrations of F-ara-ATP among several leukemia lines in vitro, which were incubated for 2 h in the presence of the indicated concentrations of F-araA using HPLC apparatus [25]. In all cell lines, the intracellular concentration of F-ara-ATP increased in a concentration-dependent manner in vitro.…”

Section: Measurement Of the Intracellular Concentration Of F-ara-atp mentioning

confidence: 99%

Synergistic effects of combination with fludarabine and carboplatin depend on fludarabine-mediated inhibition of enhanced nucleotide excision repair in leukemia

et al. 2011

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Overcoming drug resistance remains a major obstacle to curing relapsed or refractory lymphoma and obtaining a beneficial long-term prognosis for patients, despite the introduction of several salvage regimens to date. Our ultimate purpose is to establish a standard second-line salvage chemotherapy regimen for curing relapsed/refractory lymphoma. In this basic pre-clinical study, we evaluated a combination regimen consisting of 9-β-D: -arabinofuranosyl-2-fluoroadenine (F-araA) and carboplatin that targeted nucleotide excision repair (NER) of DNA in five representative leukemia lineages in vitro. Isobologram analysis demonstrated that simultaneous exposure to these two drugs produced synergistic interactions in U937 and K562 cells, in which lines showed enhanced NER activity by the measurement of UV or drug-induced DNA strand break (comet assay), or quantitation of ERCC1 mRNA (RT-PCR), a key enzyme for NER. Histone γH2AX formation was synergistically induced, but no such formation was observed after exposure to either agent alone in K562 cells. In summary, we synergistically inhibited the NER activity of leukemia cells by treating them with a combination of F-araA and carboplatin, suggesting that this combinatory regimen could be used as a novel salvage therapy for refractory or drug-resistant lymphoma.

show abstract

Simple and sensitive method for quantification of fludarabine triphosphate intracellular concentration in leukemic cells using isocratic liquid chromatography

Cited by 15 publications

References 15 publications

Targeting Glucose Consumption and Autophagy in Myeloma with the Novel Nucleoside Analogue 8-Aminoadenosine

Targeting Glucose Consumption and Autophagy in Myeloma with the Novel Nucleoside Analogue 8-Aminoadenosine

A new high-performance liquid chromatography method determines low production of 9-β-D-arabinofuranosylguanine triphosphate, an active metabolite of nelarabine, in adult T-cell leukemia cells

Synergistic effects of combination with fludarabine and carboplatin depend on fludarabine-mediated inhibition of enhanced nucleotide excision repair in leukemia

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