Abstract
In this study we evaluated the possible beneficial drug- interaction between Roflumilast (BCRP inhibitor) and Methotrexate (BCRP substrate) on viability of primary squamous cell carcinoma cell line using an in vitro technique. The KB cell line was treated with Roflumilast and Methotrexate to evaluate its anticancer activity using MTT assay. Image analysis under phase contrast microscopy was performed and flow-cytometry was done to see for cell cycle arrest as a result of drug treatment. Cell viability gradually decreased with the increasing concentrations of roflumilast, methotrexate and the cytotoxic effect with the combination of roflumilast and methotrexate also increased proportionally. Phase contrast microscopy indicated characteristic features of apoptosis which was confirmed in flow cytomtery and indicated cell cycle arrest in M phase. Efflux pump mediated multidrug resistance being a common feature among all cancers, the results of our study evidence the use of combined methotrexate and roflumilast to overcome drug resistance by exploiting the fact that the former is a BCRP substrate and latter a BCRP inhibitor. By combining the two drugs, it allows optimization of therapy by dose reduction of methotrexate and roflumilast and thereby resulting in better efficacy.
Highlights
In this study we evaluated the possible beneficial drug- interaction between Roflumilast (BCRP inhibitor) and Methotrexate (BCRP substrate) on viability of primary squamous cell carcinoma cell line using an in vitro technique
Methotrexate is a standard anticancer drug used in clinical practice while roflumilast which is a drug used for COPD, in this study has shown to possess significant anticancer activity
The above results of favourable drug interaction were confirmed by Flow Cytometric analysis of Cell cycle distribution in KB Oral Cancer cell lines treated with Roflumilast and Methotrexate combination which showed maximum cell viability inhibition in M phase of cell cycle
Summary
Forming tumor cell line) was obtained from National Center Cell Science, Pune, India. Once the cell reached the confluence, various concentrations of methotrexate and roflumilast in combination and alone were added into well followed by a 24-hour incubation period. After incubation for prescribed time periods, the images were captured using phase contrast microscopy (Nikon, Japan). MTT (100μl/well; 5mg/ml) was added to each well and incubated for 4 hours. At prescribed time 300 μl/well of DMSO was added in all wells to solubilise the precipitates. Flow-cytometry for cell cycle distribution Cell line was grown and treated with IC50 values of compounds as per standard procedures for 24 hours. According to manufactures’ instruction cell cycle reagent was added, incubated in dark for 30 minutes and was analyzed using a flow cytometer (Millipore, Guava)
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