Nucleoside-catabolizing Enzymes in Mycoplasma-infected Tumor Cell Cultures Compromise the Cytostatic Activity of the Anticancer Drug Gemcitabine

Johan Vande Voorde,Suna Sabuncuoğlu,Sam Noppen,Anders Hofer,Farahnaz Ranjbarian,Steffen Fieuws,Jan Balzarini,Sandra Liekens

doi:10.1074/jbc.m114.558924

Johan Vande Voorde, Suna Sabuncuoğlu + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m114.558924

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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.

Highlights

Gemcitabine is used to treat solid tumors
Compromised Cytostatic Activity of Gemcitabine in Mycoplasma-infected Tumor Cell Cultures—A variety of human and murine cancer cell lines were infected with M. hyorhinis (Cell line.Hyor)
A short preincubation (5 min) of the mycoplasma-infected tumor cells with thymidine phosphorylase (TP)/PyNP inhibitor (TPI) before administration of [5-3H]dUrd fully restored de novo synthesis of thymidine 5Ј-monophosphate (dTMP) formation but did not affect its formation in uninfected cells (Fig. 7, A and B). These findings indicate that dUrd, being an excellent substrate for pyrimidine nucleoside phosphorylase (PyNP)-catalyzed phosphorolysis [33], is rapidly catabolized in M. hyorhinisinfected tumor cell cultures resulting in decreased concentrations of [5-3H]dUrd-derived dUMP required for de novo dTMP synthesis

Summary

Background

Gemcitabine is used to treat solid tumors. Some mycoplasmas preferentially colonize tumors in patients. 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. As mentioned previously, phosphorylated dFdC metabolites exhibit several self-potentiating effects including inhibition of ribonucleotide reductase and CTP synthetase by dFdCDP and dFdCTP, respectively [7, 8] This results in decreased CTP and dCTP levels creating a competitive advantage for enzymatic drug activation and incorporation of dFdC in nucleic acids [6]. We investigated the pharmacological basis for these findings and identified a tandem mechanism of drug inactivation in which two catabolic mycoplasma enzymes act in concert to reduce the biological activity of dFdC in vitro and in vivo

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