Abstract
The role of the calmodulin inhibitor trifluoperazine (TFP) in modulating the cellular levels and cytotoxicity in vitro and antitumour effects in vivo of doxorubicin (DOX), was evaluated in progressively DOX-resistant (5- to 40-fold) sublines of B16-BL6 mouse melanoma. In parental-sensitive B16-BL6 cells treated for 3 h, the IC50 of DOX was 0.1 microgram ml-1, and a less than 2-fold enhancement in DOX cell kill in the presence of a noncytotoxic concentration of 5 microM TFP was observed. However, in the DOX-resistant sublines, the IC50 was 0.7 to 5.0 micrograms ml-1 DOX in the absence of 5 microM TFP and 0.3 to 0.7 microgram ml-1 DOX in the presence of 5 microM TFP. The 2- to 7.5-fold decrease in the IC50 of DOX in the presence of 5 microM TFP, was dependent on the level of DOX-resistance in the various sublines. Compared to parental-sensitive cells, a 2-fold decrease in DOX-accumulation was evident only in the 40-fold DOX-resistant subline. Further, maximal enhancement (50%) of cellular DOX accumulation in the presence of 5 microM TFP was observed only in the 40-fold resistant cells treated with 5.0 micrograms ml-1 DOX. Retention of DOX in the 40-fold resistant subline was only 20% lower than similarly treated sensitive cells, and the inclusion of TFP increased DOX retention less than 10-15%. Antitumour studies in mice with experimental pulmonary metastases revealed that although DOX and DOX plus TFP had similar antitumour activity with the parental sensitive B16-BL6 cells, the combination of DOX plus TFP was significantly more effective than DOX alone with the DOX-resistant sublines. No overt toxicity was observed in normal mice treated with doses of TFP, DOX or DOX plus TFP used for in vivo chemotherapy studies. Results from this study suggest that gross cellular DOX levels do not appear to correlate with the magnitude of resistance, and the effects of TFP in modulating DOX resistance is possibly due to mechanisms other than mere alterations in cellular drug accumulation and/or retention.
Highlights
We have recently developed progressively DOX-resistant (5- to 40-fold) B16-BL6 mouse melanoma cells with the multidrug resistant phenotype (Ganapathi, et al, 1987) and using this model system, in the present study, we have determined the characteristics of DOX cellular pharmacokinetics and cytotoxicity in vitro, antitumour activity of DOX in vivo, and the role of TFP in modulating these effects
The parental sensitive and DOX-resistant sublines were maintained as in vitro monolayer cultures at 37C in a humidified 5% CO2 plus 95% air atmosphere using Eagle's minimum essential medium (E-MEM) with Hanks salts supplemented with nonessential amino acids, sodium pyruvate, vitamins, 2 mm Lglutamine and 5% foetal bovine serum (FBS)
Log phase cultures of B16-BL6/S and B16-BL6/DOXO.25 cells harvested from monolayer cultures were resuspended in supplemented E-MEM with 5% FBS and pretreated for 1 h at 37°C. in a humidified atmosphere of 5% CO2 plus 95% air with 1 pg ml - DOX in the absence or presence of 5 pM TFP
Summary
The isolation of progressively DOX-resistant B16-BL6 mouse melanoma cells adapted to grow in vitro, in the presence of 0.025, 0.05, 0.1 and 0.25 jgml- 1 DOX, and identified as B 16-BL6/DOXO., B1 6-BL6/DOXO., B 16-BL6/DOXO. and B16-BL6/DOXO. respectively has been previously described (Ganapathi, et al, 1987). The various DOX-resistant sublines B16-BL6/DOX0.025, B 16-BL6/DOXO., B 16-BL6/ DOXO. and B16-BL6/DOXO. were approximately 5-, 10-, 20- and 40-fold resistant respectively compared to parental sensitive B16-BL6 cells (B16-BL6/S). The parental sensitive and DOX-resistant sublines were maintained as in vitro monolayer cultures at 37C in a humidified 5% CO2 plus 95% air atmosphere using Eagle's minimum essential medium (E-MEM) with Hanks salts supplemented with nonessential amino acids, sodium pyruvate, vitamins, 2 mm Lglutamine and 5% foetal bovine serum (FBS). The doubling time in vitro of parental sensitive and progressively DOX-resistant sublines of B16BL6 melanoma cells was 16-18 h. Trifluoperazine was a gift of Dr Carl Kaiser, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
