Calcitriol Reverses Induced Expression of Efflux Proteins and Potentiates Cytotoxic Activity of Gemcitabine in Capan-2 Pancreatic Cancer Cells

Efflux and influx proteins play a major role in chemo-resistance by affecting the net cellular uptake of anti-cancer drugs. Hence, alteration of the efflux and influx protein expression may result in variations of chemotherapeutics uptake and consequently cell death rate. The present study investigated the effects of pre-treatment of capan-2 pancreatic cancer cells with calcitriol, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) or silibinin on the induction of three major efflux proteins and the main gemcitabine influx protein. The influence of the pre-treatments on the net cellular uptake of gemcitabine, total ATPase activity, and cell death rate were also evaluated. Capan-2 pancreatic cancer cells were pre-treated for 24 h with calcitriol, BHT, BHA, or silibinin, followed by gemcitabine treatment. The concentration of gemcitabine was quantified using ultra-performance liquid chromatography (UPLC). Real-time polymerase chain reaction (RT-PCR) was utilized in order to investigate the expression of the mRNAs. The expression of the proteins was assessed using western blotting. Measurement of the ATPase activity was conducted utilizing a colorimetric method and viability of the cells was determined using a luminescent cell viability assay. Protein expression studies showed that BHT, silibinin, and BHA increased expression of the efflux proteins and decreased the overall uptake of gemcitabine, whereas calcitriol significantly inhibited expression of the efflux proteins and increased gemcitabine uptake. Expression of specific mRNAs correlated reasonably well with the levels of corresponding proteins. Additionally, the expression of efflux proteins and ATPase activity were well correlated, signifying that the induced efflux proteins are functionally active. Moreover, pre-treatment with calcitriol resulted in a significant increase in cell death with gemcitabine treatment, whereas, BHA significantly reduced the cell death rate. On the other hand, pre-treatment with BHT and silibinin had no significant effect on the cell death rate. Pre-treatment of the pancreatic cancer cells with calcitriol significantly increased gemcitabine cellular uptake and consequently decreased cell viability after treatment with gemcitabine, whereas BHA significantly reduced gemcitabine uptake and decreased cell death rate, which were at least partially attributed to the alteration of expression of efflux and influx proteins. This article is open to POST-PUBLICATION REVIE