Nucleoside-catabolizing enzymes in mycoplasma-infected tumor cell cultures compromise the cytostatic activity of the anticancer drug gemcitabine

Abstract

The intracellular metabolism and cytostatic activity of the anticancer drug gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdC) was severely compromised in Mycoplasma hyorhinis-infected tumor cell cultures. Pronounced deamination of dFdC to its less cytostatic metabolite 2',2'-difluoro-2'-deoxyuridine was observed, both in cell extracts and spent culture medium (i.e. tumor cell-free but mycoplasma-containing) of mycoplasma-infected tumor cells. This indicates that the decreased antiproliferative activity of dFdC in such cells is attributed to a mycoplasma cytidine deaminase causing rapid drug catabolism. Indeed, the cytostatic activity of gemcitabine could be restored by the co-administration of tetrahydrouridine (a potent cytidine deaminase inhibitor). Additionally, mycoplasma-derived pyrimidine nucleoside phosphorylase (PyNP) activity indirectly potentiated deamination of dFdC: the natural pyrimidine nucleosides uridine, 2'-deoxyuridine and thymidine inhibited mycoplasma-associated dFdC deamination but were efficiently catabolized (removed) by mycoplasma PyNP. The markedly lower anabolism and related cytostatic activity of dFdC in mycoplasma-infected tumor cells was therefore also (partially) restored by a specific TP/PyNP inhibitor (TPI), or by exogenous thymidine. Consequently, no effect on the cytostatic activity of dFdC was observed in tumor cell cultures infected with a PyNP-deficient Mycoplasma pneumoniae strain. Because it has been reported that some commensal mycoplasma species (including M. hyorhinis) preferentially colonize tumor tissue in cancer patients, our findings suggest that the presence of mycoplasmas in the tumor microenvironment could be a limiting factor for the anticancer efficiency of dFdC-based chemotherapy. Accordingly, a significantly decreased antitumor effect of dFdC was observed in mice bearing M. hyorhinis-infected murine mammary FM3A tumors compared with uninfected tumors.

Keywords: Anticancer Drug; Cancer Therapy; Cytidine Deaminase; Gemcitabine; Mycoplasma; Mycoplasma hyorhinis; Nucleoside Nucleotide Analogs; Nucleoside Nucleotide Metabolism; Phosphorylase; Pyrimidine Nucleoside Phosphorylase.

FIGURE 1.

Schematic representation of the metabolism and antimetabolic effects of dFdC. Dashed lines represent inhibitory activity. RR, ribonucleotide reductase.

FIGURE 2.

Deamination of dFdC by mycoplasma enzymes. Deamination of dFdC in the spent culture medium (A) or cell extracts (B) of confluent MCF-7 and MCF-7.Hyor cell cultures, in the presence or absence of THU (1 mm) or TPI (10 μm). The data are the mean of two independent experiments (± S.E.) and were analyzed using a t test.

FIGURE 3.

Inhibition of mycoplasma-associated dFdC deamination by natural pyrimidine nucleosides. Percentage inhibition of dFdC deamination (5 μm) by different concentrations of dThd, dUrd, or Urd. Uracil (100 μm) is used as a negative control. The data are the mean of at least two independent experiments (± S.E.). A significant correlation was found between dFdC deamination and concentration of exogenous dThd, dUrd, and Urd. (Spearman correlations for: dThd (r = 0.94; p < 0.001), dUrd (r = 0.92; p < 0.001), and Urd (r = 0.96; p < 0.001).).

FIGURE 4.

Metabolic activation of dFdC in MCF-7 and MCF-7.Hyor cells in the presence/absence of THU. Concentrations of intracellular dFdC metabolites after 24 h incubation of MCF-7 and MCF-7.Hyor cells in the presence of 0.1 μm dFdC. Panel A, dFdC. Panel B, dFdC + 1 mm THU. The data are the mean of at least two independent experiments (± S.E.). A t test was performed on the log-transformed data (*, p < 0.01).

FIGURE 5.

Metabolic activation of dFdC in MCF-7 and MCF-7.Hyor cells in the presence/absence of TPI or dThd. Concentrations of intracellular dFdC metabolites after 48 h incubation of MCF-7 and MCF-7.Hyor cells in the presence of 0.1 μm dFdC. Panel A, dFdC. Panel B, dFdC + 10 μm TPI. Panel C, dFdC + 100 μm dThd; Panel D, dFdC + 500 μm dThd. The data are the mean of at least two independent experiments (± S.E.). A t test was performed on the log-transformed data (*, p < 0.05; **, p < 0.001).

FIGURE 6.

Schematic representation of dTMP biosynthesis from [5-³H]dUrd and [5-³H]dCyd. 1, dThd kinase (cytosolic TK-1); 2, dThd kinase (mitochondrial TK-2); 3, dCyd kinase; 4, 5′-nucleotidase; 5, CMP/dCMP deaminase; 6, Cyd deaminase; 7, thymidylate synthase; 8, dTMP kinase; 9, nucleoside diphosphate kinase; 10, dUTP diphosphatase.

FIGURE 7.

De novo synthesis of dTMP in mycoplasma-infected and uninfected tumor cell cultures. Time-dependent synthesis of dTMP was measured as tritium release from [5-³H]dUrd (A and B) or [5-³H]dCyd (C and D) in the absence or presence of 10 μm TPI in CEM and CEM.Hyor cells (A and C), and L1210 and L1210.Hyor cells (B and D). The data are the mean of two independent experiments (± S.E.). A t test was performed on the log-transformed data.

FIGURE 8.

Effect of mycoplasma infection on the antitumor activity of dFdC and 5-FdUrd. In vivo antitumor activity of dFdC (panel A) and 5-FdUrd (panel B) against uninfected and M. hyorhinis-infected mammary FM3A tumors in SCID mice as represented by the tumor weight at the end of the experiment. The data represent the mean ± S.E. The interaction between infection and chemotherapy is significant for dFdC (p = 0.025) and 5-FdUrd (p = 0.0004).