Effects of amiodarone, cyclosporin A, and PSC 833 on the cytotoxicity of mitoxantrone, doxorubicin, and vincristine in non-P-glycoprotein human small cell lung cancer cell lines

Abstract

The multidrug resistance (MDR) modulators amiodarone (AM), cyclosporin A (CsA), and PSC 833 were tested for their potential to modulate cytotoxicity of doxorubicin (DOX), vincristine (VCR), and mitoxantrone (MX) in a sensitive human small cell lung carcinoma cell line GLC4, in its DOX-resistant non-P-glycoprotein subline GLC4-Adr, and in its cisplatin-resistant subline GLC4-CDDP. GLC4-Adr, in which overexpression of the so-called multidrug resistance-associated protein has been demonstrated, is 91-fold resistant for DOX, 22-fold for VCR, and 7.5-fold for MX, compared with its sensitive cell line. AM previously modulated DOX and VCR resistance in the P-glycoprotein-positive human colon cancer cell line COLO 320. Cytotoxicity was studied in the microtiter well tetrazolium assay. In the small cell lung carcinoma cell lines described above, AM did not increase cytotoxicity of DOX, but increased VCR cytotoxicity; moreover, AM was shown to be a potent modulator of MX cytotoxicity. CsA did not potentiate DOX cytotoxicity, but, at a concentration of 4 microM, it modestly increased VCR cytotoxicity in GLC4. However, 0.8 and 4.0 microM CsA protected against MX cytotoxicity in GLC4 and GLC4-CDDP, but no effect was observed in GLC4-Adr. At the much higher ID10 concentration CsA modulated MX cytotoxicity 1.6-fold in GLC4-Adr and slightly in GLC4 and GLC4-CDDP. PSC 833, a nonimmunosuppressive CsA analogue, did not alter the cytotoxicity of DOX or MX in these cell lines, but potentiated VCR cytotoxicity in GLC4-Adr at a concentration of 0.4 microM. The modulation of MX cytotoxicity by AM and the protection by CsA was confirmed in a clonogenic assay. In the colony-forming unit granulocyte-monocyte assay, no additional MX toxicity on normal bone marrow by AM was observed. Flow cytometry of cellular MX fluorescence was performed in order to elucidate the mechanism behind the AM-induced increased MX cytotoxicity. This revealed an increase in cellular MX after 1-h incubation of MX combined with AM and an inhibition of efflux from GLC4 and GLC4-Adr; CsA and PSC 833 had no effect on MX efflux. An increase in MX-induced cleavable complexes by AM in GLC4 was observed using the K+/sodium dodecyl sulfate coprecipitation assay, but no effect of CsA was found. In conclusion, AM enhances MX and VCR cytotoxicity in these sensitive, non-P-glycoprotein DOX and cisplatin-resistant small cell lung carcinoma cell lines. It also inhibits efflux of MX and causes more MX-induced cleavable complexes.