Potent reversal of multidrug resistance by ningalins and its use in drug combinations against human colon carcinoma xenograft in nude mice

Abstract

Purpose: To evaluate the pharmacological properties and the possible therapeutic applications of a series of synthetic marine natural product analogs, ningalins (N1–N6), in terms of cytotoxicity, MDR-reversing activity, and enhancement of drug combinations with antitumor agents in vitro and in vivo. Methods: XTT assays, [³H]azidopine binding to P-glycoprotein (Pgp), cellular accumulation and efflux of labeled drugs were carried out in vitro. Drug combinations using combination index, dose-reduction index, and isobologram were performed in vitro and enhancement of efficacy in drug combinations against human colon carcinoma HCT-116 xenografts were conducted with nude mice. Results: N3 at sub-IC₅₀ cytotoxic concentration (10 μM) was capable of enhancing vinblastine (VBL) cytotoxicity toward human leukemic CCRF-CEM cells about 50,000-fold as measured by the decrease of IC₅₀ of VBL. For CCRF-CEM/VBL₁₀₀₀ (1,500-fold resistant to VBL and overexpressing Pgp), N3 and N5 enhanced VBL cytotoxicity as much as 6.2 million-fold and 210,000-fold, respectively. Moreover, N3 and N5 collaterally made CCRF-CEM/VBL₁₀₀₀ cells 4,000-fold and 130-fold, respectively, more susceptible to VBL than the parent CCRF-CEM cells. In human mammary carcinoma cells MX-1/paclitaxel which were 170-fold resistant to taxol and 38-fold resistant to VBL, N3 was capable of enhancing VBL effect as much as 6,000-fold. Combination therapy on murine P388/doxorubicin (DX) leukemia with DX + N3 or taxol + N3 achieved greater efficacy than the therapy with each drug alone. Impressively, nude mice, bearing human colon carcinoma HCT-116 cells, treated with a suboptimal dosage of taxol in combination with N3, N5 or N6 led to shrinkage of established tumor and achieved total tumor remission, while taxol alone had no tumor disappearance in this xenograft model. Furthermore, the enhancement of antitumor effect by ningalins, at least in parts, are due to inhibiting Pgp which was supported by the observation that the ningalins compete for [³H]azidopine binding to Pgp, increase the cellular accumulation of VBL or taxol, and inhibit drug efflux from the tumor cells. Conclusion: The profound enhancement of antitumor cytotoxicity of vinblastine and taxol in vitro by ningalins may have multiple mechanisms including the MDR-reversing effects. The mechanisms for collateral sensitivity by ningalins against sensitive (parent) cells are not yet clear. The marked enhancement of therapeutic effect of taxol by ningalins against xenograft tumors in nude mice suggests potential applications of therapeutic use of ningalins.