triphosphoric-acid-1-adenosin-5--yl-ester-3-(3-methylbut-3-enyl)-ester and Breast-Neoplasms

The nitrogen-containing bisphosphonate zoledronic acid (ZOL), a potent inhibitor of farnesyl pyrophosphate synthase, blocks the mevalonate pathway, leading to intracellular accumulation of isopentenyl pyrophosphate/triphosphoric acid I-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester (IPP/ApppI) mevalonate metabolites. IPP/ApppI accumulation in ZOL-treated cancer cells may be recognized by Vγ9Vδ2 T cells as tumor phosphoantigens in vitro. However, the significance of these findings in vivo remains largely unknown. In this study, we investigated the correlation between the anticancer activities of Vγ9Vδ2 T cells and the intracellular IPP/ApppI levels in ZOL-treated breast cancer cells in vitro and in vivo. We found marked differences in IPP/ApppI production among different human breast cancer cell lines post-ZOL treatment. Coculture with purified human Vγ9Vδ2 T cells led to IPP/ApppI-dependent near-complete killing of ZOL-treated breast cancer cells. In ZOL-treated mice bearing subcutaneous breast cancer xenografts, Vγ9Vδ2 T cells infiltrated and inhibited growth of tumors that produced high IPP/ApppI levels, but not those expressing low IPP/ApppI levels. Moreover, IPP/ApppI not only accumulated in cancer cells but it was also secreted, promoting Vγ9Vδ2 T-cell chemotaxis to the tumor. Without Vγ9Vδ2 T-cell expansion, ZOL did not inhibit tumor growth. These findings suggest that cancers-producing high IPP/ApppI levels after ZOL treatment are most likely to benefit from Vγ9Vδ2 T-cell-mediated immunotherapy.

Topics: Adenosine Triphosphate; Animals; Antigens, Neoplasm; Breast Neoplasms; Cell Line, Tumor; Cytotoxicity, Immunologic; Diphosphonates; Female; Hemiterpenes; Humans; Imidazoles; Mice; Mice, Inbred NOD; Mice, SCID; Organophosphorus Compounds; Phosphorylation; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocytes; Zoledronic Acid

Nitrogen-containing bisphosphonates (N-BPs) are shown to inhibit a key enzyme of intracellular mevalonate pathway, FPP synthase, leading to intracellular accumulation of pathway metabolites isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). In our previous studies we have shown that a new type of ATP analog, ApppI (triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) ester), is also formed in addition to IPP and DMAPP accumulation. ApppI has cytotoxic effects leading to direct apoptosis of various cancer cells. In this study we present a validated method based on ion-pair LC-MS(2) for the analysis of isomeric mevalonate pathway metabolites and ATP analogs in cell culture samples. Limit of quantitation for IPP and DMAPP was 0.030microM (1.35fmol on-column) and for ApppI and ApppD 0.020microM (0.9fmol on-column). Acceptable accuracies and precision were also obtained for quality control samples in low and high concentrations of the calibration curve. In addition, we present a new method for quantitation of each coeluting isomer utilizing the peak intensity ratios of two characteristic fragment ions of each compound. For IPP and DMAPP, fragment ions m/z 177 and m/z 159 in the MS(2) were monitored, whereas for ATP analogs, ApppI and ApppD (triphosphoric acid 1-adenosin-5'-yl ester 3-(3-methylbut-2-enyl) ester), the same fragments in the MS(3) spectra were followed. IPP and DMAPP accumulation as well as ApppI and ApppD formation was demonstrated using MCF-7 breast cancer cells. Cells were treated with 25muM zoledronic acid (an N-BP) for 24h, conditions found to induce significant production of the metabolites. We found that the total amount of IPP and DMAPP was 2.4nmol/mg of protein and amount of ApppI and ApppD was 1.1nmol/mg protein. Relative portions of the isomers were approximately 1:4 IPP:DMAPP and 3:7 ApppI:ApppD. Untreated control samples did not contain IPP, DMAPP, ApppI or ApppD.

Topics: Adenosine Triphosphate; Breast Neoplasms; Cell Line, Tumor; Chromatography, Liquid; Drug Stability; Hemiterpenes; Humans; Linear Models; Mevalonic Acid; Organophosphorus Compounds; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization