Activity of microcystin-LR against xenografts expressing the OATP1B1 transporter.

C63 OATP1B3 transporter gene expression has been detected in lung, gastrointestinal, hepatic and breast cancers. We hypothesized that transporter-specific cytotoxic molecules could be identified which selectively utilize OATP1B3 for cellular uptake, and which would selectively kill OATP1B3-expressing tumor cells. Previously, we have demonstrated that HeLa cells transfected to express either OATP1B3 or the closely related OATP1B1 transporters are exquisitely sensitive in vitro to microcystin-LR (MCLR) and its structural variants (Monks NR, et al., Molecular Cancer Therapeutics, 6, 587-598, 2007). Microcystins are naturally occurring cyclic hepatapeptides produced by cyanobacteria, and are known hepatic toxins. The next step was to prove the principle that microcystins could treat transporter-expressing tumors in vivo without lethal toxicity. To this end, we generated stable cell lines expressing both OATP1B1 and OATP1B3 for xenograft studies. The OATP1B1 and OATP1B3 stable cell lines exhibit [3H]-BQ123 uptake characteristics similar to those reported previously in our transient transfection model. MCLR in vitro cytoxicity was also comparable to the transient model, with IC50s of 19.5 and 1.5nM for OATP1B3 and OATP1B1, respectively. In our previous studies other microcystin structural variants were 10-fold more potent than MCLR in OATP1B1-expressing cells, and 50-fold more potent than MCLR in OATP1B3-expressing cells. Because of issues of expense and availability, MCLR was the microcystin used for the first xenograft experiments, and therefore the chosen xenograft model was OATP1B1, since cells expressing this transporter are >10-fold more sensitive to MCLR than the OATP1B3 cells. To validate the model in vivo, real-time PCR analysis of OATP1B1 transporter expression in the xenografts demonstrated that the transporter mRNA levels of OATP1B1 were preserved during in vivo tumor growth compared to the in vitro levels. To test the efficacy of MCLR against OATP1B1 xenografts, cohorts of 4-6 weeks old Female Athymic nude mice (Nu/Nu) were subcutaneously implanted with 5x106 cells. Once the tumors were palpable, the mice were injected IP with a sub-LD50 concentration of MCLR (35μg/kg) every 48 hours for 18 days (10 doses of MCLR); body weight and tumor size were measured every second day. Each cohort contained 3-5 mice and 2 independent experiments were performed. By day 8 the MCLR-treated tumor volume was 40% that of the control tumors, and by day 16 the MCLR-treated tumor volume was 49% of control tumor volume. One of 8 mice in the 2 experiments died during the experimental period in the treated group and none of 9 mice died in the control group. The MCLR-treated group body weight ended at the baseline weight, while the control group gained approximately 15% over the experimental period. Pathological assessment of liver sections following the treatment is in progress. In summary, we have demonstrated that microcystins can be used to inhibit in vivo tumor growth in xenografts expressing a transporter which facilitates their intracellular uptake. Further studies will screen microcystin structural variants, both natural and synthetic, to identify a suitable analog which can be used to treat OATP1B3-expressing tumors.