Mycoplasma hyorhinis-encoded cytidine deaminase efficiently inactivates cytosine-based anticancer drugs

Abstract

Mycoplasmas may colonize tumor tissue in patients. The cytostatic activity of gemcitabine was dramatically decreased in Mycoplasma hyorhinis-infected tumor cell cultures compared with non-infected tumor cell cultures. This mycoplasma-driven drug deamination could be prevented by exogenous administration of the cytidine deaminase (CDA) inhibitor tetrahydrouridine, but also by the natural nucleosides or by a purine nucleoside phosphorylase inhibitor. The M. hyorhinis-encoded CDAHyor gene was cloned, expressed as a recombinant protein and purified. CDAHyor was found to be more catalytically active than its human equivalent and efficiently deaminates (inactivates) cytosine-based anticancer drugs. CDAHyor expression at the tumor site may result in selective drug inactivation and suboptimal therapeutic efficiency.

Keywords: (d)Ado, (2′-deoxy)adenosine; (d)Guo, (2′-deoxy)guanosine; (d)Ino, (2′-deoxy)inosine; (d)Urd, (2′-deoxy)uridine; 3TC, 2′,3′-dideoxy-3′-thiacytidine; CDA, cytidine deaminase; Cancer; Cytidine deaminase; Gemcitabine; Imm-H, Immucillin-H; Mycoplasma; NA, nucleoside analogue; Nucleoside analogue; PNP, purine nucleoside phosphorylase; Purine nucleoside phosphorylase; ara-Cyd, cytosine arabinoside; dFdC, gemcitabine; dFdU, 2′,2′-difluoro-2′-deoxyuridine; dThd, thymidine; ddC, 2′,3′-dideoxycytidine.

Fig. 1

Molecular structure of the (2′-deoxy)cytidine analogues gemcitabine (A) and cytarabine (B).

Fig. 2

Purity evaluation of the CDA_Hyor-GST fusion protein. Three different concentrations (i.e. 10, 1 and 0.1 μg) of the purified enzyme preparation were analyzed using SDS–PAGE. Proteins were stained using Bio-Safe™ Coomassie G-250 Stain (Bio-Rad Laboratories, CA, USA).

Fig. 3

Inhibition of mycoplasma-associated [5-³H]dFdU formation by natural nucleosides. Formation of [5-³H]dFdU from [5-³H]dFdC in the tumor cell-free supernatant of mycoplasma-infected and control MDA.MB.231 (A) and MCF-7 (B) breast cancer cell cultures in the presence/absence of different concentrations of pyrimidine (dThd and Urd) and purine (Ado and Ino) nucleosides. The data are the mean of at least two independent experiments (±S.E.M.).

Fig. 4

Kinetic analysis of CDA_Hyor- and CDA_Human-catalyzed deamination of natural nucleosides and nucleoside analogues Deamination of different concentrations of Cyd (A and B), dCyd (C and D), dFdC (E and F) and ara-Cyd (G and H) by CDA_Hyor (A, C, E and G) or CDA_Human (B, D, F and H). The data are the mean of at least two independent experiments (±S.E.M.).