Biochemical modulation has played an important role in the development of
cancer chemotherapy. The combined effects of
theanine, a specific
amino acid in
green tea, and
glutamate transporter inhibitors on the antitumor activity of
doxorubicin (DOX), were investigated and we clarified the biochemical mechanisms of action of these modulators. In M5076 ovarian
sarcoma-bearing mice,
theanine significantly enhanced the inhibitory effect of DOX on
tumor growth and increased the DOX concentration in the
tumor, compared to DOX-alone group. Furthermore, the
oral administration of
theanine or
green tea similarly enhanced the antitumor activity of DOX. Moreover, the combination of
theanine with DOX suppressed the hepatic
metastasis of ovarian
sarcoma. In contrast, an increase in DOX concentration was not observed in normal tissues, such as liver and heart. Namely,
theanine did not enhance, rather it tended to normalize the increase of
lipid peroxide (LPO) levels and reduction of
glutathione peroxidase activity as indicators of the DOX-induced side toxicity. On the other hand, in vitro experiments proved that
theanine inhibited the efflux of DOX from
tumor cells, supporting a
theanine-induced increase in the DOX concentration in
tumors in vivo. Moreover,
theanine significantly inhibited the
glutamate uptake by M5076 cells similar to specific inhibitors. Two astrocytic high-affinity
glutamate transporters, GLAST and GLT-1, were expressed in M5076 cells. These results suggested that the inhibition of DOX efflux was induced by
theanine-mediated inhibition of
glutamate transporters. The reduction in the concentration of
glutamate in
tumor cells caused by
theanine induced decreases in the intracellular
glutathione (GSH) and GS-DOX conjugate levels. As the expression of MRP5 in M5076 cells was confirmed, it is suggested that the GS-DOX conjugate was transported extracellularly via the MRP5/
GS-X pump in M5076 cells and that
theanine affected this route. Namely,
theanine increases the concentration of DOX in a
tumor in vivo through inhibition of the
glutamate transporter via the
GS-X pump. Similarly,
dihydrokainate (DHK) and
L-serine-O-sulfate (SOS), specific
glutamate transporter inhibitors, indicated the enhancement of the DOX antitumor activity via inhibition of
glutamate uptake. Therefore, we revealed the novel mechanism of enhancement of antitumor efficacy of DOX via the inhibition of
glutamate transporters. Similarly,
theanine enhanced the antitumor activities of other
anthracyclines,
cisplatin and
irinotecan. Consequently, the modulating effect of
theanine on the efficacy of
antitumor agents is expected to be applicable in clinical
cancer chemotherapy.