The effect of PKR kinase activation on the sensitivity of choriocarcinoma cells to a chemotherapeutic agent

Objective: Objective: The aim of this study was to determine how the proliferation and apoptotic properties of choriocarcinoma (swan 71) cells grown in cell culture would change as a result of PKR activation. In addition, it was aimed to determine the sensitivity of PKR activation to the chemotherapeutic agent doxorubicin in choriocarcinoma cells and to show that it can be an alternative that can be used in cancer treatment. Materials and Methods: First, human placenta trophoblast carcinoma swan 71 cells were grown by cell culture method. Cells were divided into two groups as doxorubicin group and poly I:C/LPS/doxorubicin group. In the doxorubicin group, different doses of doxorubicin (0.39 µg/ml, 1.56 µg/ml, 6.25 µg/ml, 25 µg/ml, 100 µg/ml) were applied to the cells for 24 hours. To induce PKR activation, LPS and poly I:C were applied to swan 71 cells for 48 hours. Then the groups; control, doxorubicin, poly I:C/LPS and poly I:C/LPS/doxorubicin. According to the experimental groups, 50 μl of poly I:C and 500 μl of LPS applied to the cells were incubated for 48 hours, and 100 μl of doxorubucin for 24 hours. MTT viability assay was performed to determine the effects of LPS, poly I:C and doxorubicin on swan 71 cells. Then, viability, necrosis and apoptosis pathways were examined by flow cytometry in cells treated with doxorubicin for 24 and 48 hours and in cells treated with poly I:C/LPS for 48 and 72 hours. Finally, experimental groups were treated with doxorubicin for 24 hours and poly I:C/LPS for 48 hours, and protein was obtained from these cells by protein isolation method. Then, the amount of protein obtained by the bradford experiment was determined. In order to detect PKR activation and to determine the effect of doxorubicin on PKR activation, western blot experiment was performed and p-PKR and α-tubulin were examined. Results: Doxorubicin in swan 71 cells treated with different doses (0.39 µg/ml, 1.56 µg/ml, 6.25 µg/ml, 25 µg/ml, 100 µg/ml) in the experiment performed with the cell counter, compared to the control Statistically significant decreases were observed in percent viability as the concentration increased (p < 0.05, p < 0.001). Statistically significant dose- and time-dependent reductions were observed in PKR-activated cells treated with different doses of doxorubicin compared to control (p < 0.05, p < 0.001). Doxorubicin provided dose- and time-dependent resistance to the lethal effect in PKR-activated cells and was found to be statistically significant. In the following, MTT test; were evaluated in the control, LPS/poly I:C, doxorubicin and LPS/poly I:C/dox groups, but no results were obtained. When the 24-hour and 48-hour viability values in the control, LPS/poly I:C, doxorubicin and LPS/poly I:C/dox groups were examined in flow cytometry, a significant increase was observed in the poly I:C/LPS group compared to the control (p<0.05, p<0.001). A statistically significant increase was found in the Poly I:C/LPS/dox group compared to the doxorubicin group (p<0.05, p<0.001). Then, when the necrosis pathway was examined for 24 and 48 hours in the control, LPS/polyI:C, doxorubicin and LPS/poly I:C/dox groups, it was observed that the LPS/polyI:C group did not go to the necrosis pathway compared to the control, the LPS/polyI:C/dox group On the other hand, it was observed that doxorubicin went to the necrosis pathway according to the group. When the apoptosis pathways were examined for 24 hours and 48 hours in the control, LPS/polyI:C, doxorubicin and LPS/poly I:C/dox groups, it was observed that the LPS/polyI:C/dox group went into apoptosis compared to the doxorubicin group. Finally, p-PKR activation was detected in all groups by western blot analysis. It was observed that PKR activation inhibited the sensitivity of doxorubicin. Conclusion: In the current study, it was observed that PKR activation showed resistance to doxorubicin, a chemotherapeutic agent, in choriocarcinoma cells.