Gemcitabine and Thapsigargin Counteract the Effects of 5‐Fluorouracil on Nuclear Transport during Apoptosis through Different Mechanisms of Action

Abstract

Regulation of nuclear transport is an essential component of apoptosis. As chemotherapy induced cell death progresses, nuclear transport and the nuclear pore complex (NPC) are disrupted and dismantled. Specifically, 5‐flurouracil (5‐FU) increases NPC permeability and disrupts the Ran gradient, decreasing it's nuclear levels and inhibiting nuclear export. As a result, combination of 5‐FU with other chemotherapeutics, such as topotican, increases their efficiency, by retaining pro‐apoptotic factors in the nucleus. Here we examine the ability of 5‐FU to enhance the effects of another drug, gemcitabine, previously shown to work synergistically with nuclear transport inhibitors. Interestingly, we found that while both 5‐FU and gemcitabine cause a decrease in nuclear Ran, combination of the drugs led to no significant change in Ran localization. In order to determine the mechanism of action for both drugs and the means of their counteraction, we explored their effects on NPC permeability. As expected, 5‐FU increased NPC permeability; however, gemcitabine did not disrupt the NPC. Additionally, gemcitabine counteracted 5‐FU, indicating that their antagonistic effects on Ran localization occur at the level of NPC permeability. As 5‐FU's effects on the NPC are known to be calcium dependent, we also explored whether gemcitabine similarly counteracts thapsigargin, an inhibitor of ER calcium uptake and known disruptor of nuclear Ran localization. Interestingly, gemcitabine had no effect on thapsigargin's ability to decrease nuclear Ran levels. Furthermore, thapsigargin had no effect on NPC permeability. Instead, thapsigargin counteracted the ability of 5‐FU to decrease nuclear Ran levels, despite the fact that it was unable to change 5‐FU induced NPC permeability. These results suggest that gemcitabine and thapsigargin mediate similar negative effects on Ran and nuclear transport counteractive to 5‐FU, but thapsigargin does so via an unknown mechanism. Therefore, the effects of 5‐FU and thapsigargin may reflect various potential means of calcium mediated changes to nuclear transport in early apoptosis. Future work will involve exploring the direct impact of these treatments on calcium levels. Importantly, the impacts of all drug treatments on Ran in the nucleus were due to changes in cellular localization differences alone, as immunoblot analysis demonstrated no changes in Ran protein expression levels. The combinatory effects of these drugs on the NPC and nuclear transport were unexpected based on previous work, and indicate the wide variation in nuclear disruption in apoptosis. At this level it is unclear if the impacts of 5‐FU, thapsigargin, and gemcitabine on nuclear transport and the NPC alone will be sufficient to efficiently predict the efficacy of these combinations on apoptosis. The counteraction of gemcitabine or thapsigargin on 5‐FU at this level may or may not be a predictor of cell death, and thus our ongoing work involves studies of cell viability and the localization of apoptotic factors with these treatments. Understanding the molecular mechanisms of these drugs will help to inform the usefulness of their combination in cancer treatment, particularly in cases of drug resistance.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.