5‐Fluorouracil and Gemcitabine Synergistically Facilitate the Nuclear Accumulation of Tumor Suppressors p53 and p27, Resulting in Decreased Cell Viability

Abstract

Understanding the molecular mechanism of action for chemotherapeutic drugs can help improve combinatorial therapies, particularly of existing treatments known to develop resistance. 5‐fluorouracil (5‐FU) is known to promote apoptosis in cancer cells in part by increasing nuclear pore complex (NPC) permeability and disrupting the Ran gradient, a key regulator of nuclear transport. Therefore, combination of 5‐FU with other drugs that are impacted by excess nuclear export of target proteins can potentially improve their effectiveness. For example, previous work has shown that 5‐FU's impact on nuclear export increases the effectiveness of topotecan by increasing the nuclear accumulation of topoisomerase and p53. The chemotherapy gemcitabine is also known to work synergistically with nuclear export inhibitors, therefore we sought to determine its effects in combination with 5‐FU. Specifically, we analyzed the effects of 5‐FU and gemcitabine on the nuclear localization of the tumor suppressor proteins p27 and p53 in HeLa cells. For p27, all drug treatments caused a decrease in nuclear localization; however, p27 protein expression was also reduced suggesting that lower nuclear levels may be due to less p27 available in the cells. Yet, the decrease in nuclear localization was not always comparable to the reduction in available protein, suggesting some level of nuclear export is blocked despite nuclear protein levels being lower than the control cells. This effect is more evident for p53, where 5‐FU increased its nuclear localization over that of the control, despite decreasing overall p53 expression. Furthermore, drastic reduction in p53 expression caused by gemcitabine either alone or in combination with 5‐FU did not translate to massive impacts on nuclear localization. As such, while 5‐FU and gemcitabine alone decreased expression of key regulators of cell cycle arrest and apoptosis, these drugs also led to an increased likelihood of their nuclear accumulation which persisted when the drugs were combined. These effects on the nuclear export of p27 and p53 also translated to impacts on cell viability, where the nuclear accumulation of these tumor suppressors mirrored increases in cell death. While each drug alone had minor effects on both nuclear protein accumulation (2‐fold increase) and cell viability (30–50% reduction), the combination of 5‐FU and gemcitabine caused a ~9‐fold increase in nuclear accumulation of p27 and p53, which was reflected in a 70% reduction in cell viability. These results indicate that the decreased expression of tumor suppressor proteins in gemcitabine treated cells can in part be overcome by increasing their nuclear localization via 5‐FU treatment. Using these drugs in combination can effectively lead to induction of apoptosis and thus suggests a potential mechanism for overcoming gemcitabine 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.