Tomomi Sugiyama

Efficacies of tea components on doxorubicin induced antitumor activity and reversal of multidrug resistance.

Considering of novel biochemical modulation by some foods and beverages, we have performed screening for green tea components that have enhancing effects on doxorubicin (DOX) induced antitumor activity. Components, such as caffeine, theanine, (-)-epigallocatechin gallate (EGCG) and flavonoids have inhibitory effects on the DOX efflux from Ehrlich ascites carcinoma cells. Thus, it is suggested that EGCG and flavonoids may enhance DOX induced antitumor activity and increase the DOX concentrations in tumors through the inhibition of DOX efflux. It is expected that these components in green tea exhibit low toxicity and that there are few side effects of drinking green tea in combination with an antitumor agent. We think that the intake of a favorite beverage favors a positive mental attitude of a patient and increases the efficacy of the chemotherapeutic index, and that this efficacy is useful for improving the quality of life on cancer chemotherapy. In DOX resistant P388 leukemia cell bearing mice theanine increased the DOX induced efficacy through an increase in the DOX concentrations in the tumors. Theanine attacked the same transport process for DOX in both types of cells, elevated the DOX concentration and increased the DOX induced antitumor activity.

Protective effect of flavonoids on doxorubicin-induced cardiotoxicity

We have examined the effect of alpha G-Rutin and luteolin on doxorubicin (DOX) toxicity in mice. In the heart, the lipid peroxide level, increased to 1.5 times of the normal level induced by DOX, decreased to the normal level after treatment with alpha G-Rutin or luteolin (i.p.). Glutathione peroxidase (GSHpx) activity, decreased to 73% of normal activity after DOX treatment, was shown to recover by the combined flavonoids. The lipid peroxide level in bone marrow cells increased to 5.9 times of the normal level by DOX treatment, whereas this level in the extra bone marrow cells did not change by treatment with DOX. The combination of alpha G-Rutin and luteolin with DOX significantly inhibited the DOX induced-increment of the lipid peroxide level in bone marrow cells. Flavonoids have also reduced the effect of DOX toxicity by oral administration. It is suggested that it is possible to reduce DOX toxicity by the intake of food including flavonoids. In NADPH-dependent lipid peroxidation, alpha G-Rutin and luteolin showed concentration-dependent inhibition. Therefore, we considered that the reduction effect of DOX toxicity by flavonoids was caused by antioxidative action and other effect of the flavonoids.

The effects of theanine, as a novel biochemical modulator, on the antitumor activity of adriamycin

We studied the effects of theanine, a component of green tea leaves, on the antitumor activity of adriamycin (ADR) from the biochemical modulation view point. In vitro, theanine inhibited the ADR efflux from Ehrlich ascites carcinoma cells and maintained the ADR concentration in tumor cells. Theanine enhanced the inhibitory effect of ADR on tumor growth by 2.1-fold in vivo, and increased 2.9-fold the ADR concentration in the tumor, compared to the ADR alone group. An increase in ADR concentration was not observed in normal tissues, such as the heart and liver. Theanine did not enhance, rather tended to normalize the increase of lipid peroxide level and reduction of glutathione peroxidase activity as indicators of the ADR-induced side toxicity.

Enhancing effects of green tea components on the antitumor activity of adriamycin against M5076 ovarian sarcoma

We have investigated the combined treatment of components of green tea with adriamycin against M5076 ovarian sarcoma, which exhibits low sensitivity to adriamycin. In M5076 tumor-bearing mice, the injection of adriamycin alone did not inhibit tumor growth, whereas the combination of theanine and adriamycin significantly reduced the tumor weight to 62% of the control level. When combined with theanine, effective antitumor activity of adriamycin was observed without an increase in the dosage. Theanine specifically increased the adriamycin concentration in the tumor by 2.7-fold. In contrast, theanine decreased the adriamycin concentrations in normal tissues. On the other hand, in vitro experiments proved that theanine inhibited the efflux of adriamycin from tumor cells, suggesting a theanine-induced increase in the adriamycin concentration in such tumors in vivo. Furthermore, the oral administration of theanine or green tea similarly enhanced the antitumor activity of adriamycin. In conclusion, the combination of theanine with adriamycin showed antitumor efficacy in spite of the non-effective dose of adriamycin on M5076 ovarian sarcoma. We have found that the modulating action of theanine is useful in clinical cancer chemotherapy.

Theanine and glutamate transporter inhibitors enhance the antitumor efficacy of chemotherapeutic agents.

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.

Enhancement of the activity of doxorubicin by inhibition of glutamate transporter

Theanine enhanced doxorubicin (DOX) induced antitumor activity by increasing the concentration of DOX in the tumor through the inhibition of efflux of DOX from tumor cells. As theanine reduced the level of glutamate via suppression of the glutamate transporter in tumor cells, we studied the change in the intracellular concentration of glutathione (GSH) and the correlation with the GSH S-conjugate export (GS-X) pump. The reduction in the concentration of glutamate in tumor cells caused by theanine, induced decreases in the intracellular GSH and GS-DOX levels. The expression of MRP5 in M5076 cells, was confirmed. We concluded 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.

Inhibition of glutamate transporter by theanine enhances the therapeutic efficacy of doxorubicin

Theanine, a major amino acid existing in green tea, enhanced the antitumor activity of doxorubicin (DOX) due to inhibition of DOX efflux from tumor cells. In order to clarify the mechanism, we have investigated the contribution of glutamate transporters to the action of theanine, because theanine is a glutamate analogue. In M5076 ovarian sarcoma cells, glutamate transport inhibitors reduced the efflux of DOX, as well as theanine. Incidentally, theanine significantly inhibited the glutamate uptake by M5076 cells in a concentration-dependent manner similar to specific inhibitors. These results suggested that the inhibition of DOX efflux was induced by the inhibition of glutamate transport by theanine. In addition, RT-PCR and Western blot analysis revealed the expression of GLAST and GLT-1, astrocytic high-affinity glutamate transporters, in M5076 cells. Thus, theanine was shown to competitively inhibit the glutamate uptake by acting on these glutamate transporters. This action suggested the contribution of glutamate transporters to the inhibition of DOX efflux by theanine. We revealed the novel mechanism of enhancement of the antitumor efficacy of DOX via the inhibition of glutamate transporters by theanine.

Membrane Transport and Antitumor Activity of Pirarubicin, and Comparison with Those of Doxorubicin

We have compared the membrane transport and antitumor activity of pirarubicin with those of doxorubicin in M5076 ovarian sarcoma, which exhibits low sensitivity to doxorubicin. Pirarubicin was rapidly taken up by M5076 cells and the intracellular concentration of pirarubicin reached more than 2.5‐fold that of doxorubicin. In terms of the 50% cell growth‐inhibitory concentration in vitro, pirarubicin was more effective than doxorubicin. Thus, the intracellular concentration influenced the cytotoxicity of these anthracycline agents. On comparison of the nuclear uptake of pirarubicin and doxorubicin, the nucleus/cell ratio of pirarubicin was found to be about 40%, whereas that of doxorubicin reached more than 80%. As the intranuclear concentration of pirarubicin is dependent on nuclear transport, the increases in not only cell membrane transport, but also nuclear membrane transport contributed to the enhancement of the efficacy of pirarubicin. In M5076 solid tumor‐bearing mice, pirarubicin reduced the tumor weight to 60% of the control level, although doxorubicin had no effect. These results were supported by the intracellular uptake of pirarubicin. Moreover, theanine, which inhibited the pirarubicin efflux from M5076 cells, increased by 1.3‐fold the pirarubicin concentration in the tumor and enhanced the therapeutic efficacy of pirarubicin 1.7‐fold. In conclusion, our results suggest that an increase in the concentration of an anthracycline derivative in tumor cells due to alteration of cell membrane transport results in enhancement of the antitumor activity.

Improvement of idarubicin induced antitumor activity and bone marrow suppression by theanine, a component of tea

We have examined the effect of theanine, a specific amino acid in green tea, on idarubicin (IDA)-induced antitumor activity and toxicity. In combination with theanine, IDA (0.25 mg/kg per day x4 days, a dose that does not show antitumor activity) had significant antitumor activity in P388-bearing mice. The IDA concentration in the tumors in the theanine plus IDA group increased to twice the level in the IDA alone group. Furthermore, the decrease in tumor weight caused by IDA at 1.0 mg/kg per day x4 days (at this dose IDA exhibits antitumor activity) was significantly amplified by theanine. The numbers of leukocyte and bone marrow cells decreased significantly on IDA injection. Theanine significantly reversed these changes. These results suggest that theanine selectively moderates the IDA-induced toxicities. Until recently, the antitumor activity and related toxicities of this chemotherapeutic agent in leukemia could not be distinguished. Theanine increases the IDA-induced antitumor activity and ameliorates the toxicities.

Effect of dihydrokainate on the antitumor activity of doxorubicin

For biochemical modulation, components of green tea have been shown to be useful modulators in combination with doxorubicin (DOX). We have confirmed that theanine enhances the antitumor activity of DOX due to inhibition of DOX efflux from tumor cells. Because theanine is a glutamate analogue, we found that it is associated with a change in the drug transport system on the tumor cell membrane, in particular glutamate transporters. We examined the effect of dihydrokainate (DHK), one of the useful glutamate transporter inhibitors. DHK also inhibits DOX efflux significantly and reduces the glutamate uptake by Ehrlich ascites carcinoma cells. The potential contribution of glutamate transporters not only to glutamate uptake but also to cell membrane export of DOX has been shown. In addition, the combination of DHK with DOX significantly enhances the antitumor activity of DOX, by 1.8-fold (P<0.001). The DOX concentration in tumors significantly increases on combination with DHK and is correlated with the reduced tumor weight. On the other hand, DHK tends to reduce the DOX concentration in normal tissues. We expect that DHK has different actions in tumor and normal tissues because different isoforms of glutamate transporters are expressed in the two tissues. Thus, the results suggest that DHK is a novel and useful modulator for inducing enhancement of antitumor activity.