Levels of viral glycoprotein reflect enhanced effectiveness of combined drug treatments

Abstract

A chemosensitivity assay with small replicate Mm5mt/cl C3H mammary tumor cell cultures was developed to determine whether charges in viral antigen expression and release into culture fluids could be utilized as an in vitro measure of single and combined drug effect. The measurement of drug concentrations required for identical 50% reductions in viral antigen levels (ED 50s) allowed the dosage-dependence of the same drug to be compared alone and in combination drug treatments. The 52,000 MW viral envelope glycoprotein (gp52) of the mouse mammary tumor virus (MMTV) was measured in culture fluids of control and drug-treated cultures while cell density was simultaneously determined by cell staining and OD 664 mu determination. While extracellular gp52 levels and cell density increased over 72 hours in control cultures, dose-dependent declines in both parameters provided dual measures of effect for combination CAF treatment [cyclophosphamide: doxorubicin (Adriamycin): 5-fluorouracil], combination CMF treatment [cyclophosphamide: methotrexate: 5-fluorouracil] and single component treatments of these combinations. Comparison of ED 50s for gp52 reduction revealed that each drug's ED 50 concentration was lower in combination treatment than it was in single treatment. In combined drug treatments, the ED 50 of doxorubicin was lowered from 5.17 μM to 0.36 μM, that of 5-fluorouracil was lowered from 0.69 μM to 0.22 μM and that of methotrexate was lowered from 15 nM to 1.65 nM. Reductions of ED 50 in combined drug treatments argued for drug synergisms in these combinations. When drug concentrations, rather than gp52 reductions, were fixed at identical levels in single and combined treatments, additional declines in gp52 levels reflected the enhanced effects of CAF and CMF combinations over single drug treatments. When the anti-estrogen, tamoxifen, was added to CAF and CMF treatments, further declines in gp52 levels (below CAF and CMF levels) reflected the fact that these hormone-augmented treatments were more effective than either hormone or combination chemotherapy alone. These results indicate that decreases in extracellular levels of MMTV gp52 can be utilized in this in vitro assay an an alternative measure of chemotherapeutic effect for evaluating the synergistic of antagonistic nature of drug or hormone interactions and as a tool for in vitro optimization of combined drug effect.