Nrf2-siRNA Enhanced the Anti-Tumor Effects of As(2)O(3) in 5-Fluorouracil-Resistant Hepatocellular Carcinoma by Inhibiting HIF-1α/HSP70 Signaling

PURPOSE: Chemoresistance is a major factor contributing to the failure of cancer treatment. The conventional chemotherapy agent 5-fluorouracil (5-FU) has been used for cancer treatment for decades. However, its use is limited in the treatment of hepatocellular carcinoma (HCC) due to acquired resistance. Nrf2 (NF-E2-related factor 2) is known to be associated with drug resistance across a wide range of cancer types. Also, since arsenic trioxide (As(2)O(3)) showed antitumor effects on HCC, the purpose of this study was to determine whether As(2)O(3) and Nrf2-siRNA could inhibit HCC synergistically. METHODS: We generated two separate 5-FU-resistant HCC cell lines (SNU-387/5-FU and Hep3B/5-FU). Western blotting was used to determine protein levels. An efficient lentiviral delivery system was used to establish stable knockdown or overexpression of Nrf2 and HIF-1α. In vitro and in vivo analyses of the effects of Nrf2 gene knockdown and As(2)O(3) on 5-FU-resistant HCC cells were conducted. RESULTS: The expression of Nrf2 was higher in the 5-FU-resistant HCC cell lines than in the parental cell lines. When coupled with Nrf2 knockdown, As(2)O(3) treatment significantly decreased 5-FU-resistant SNU-387 and Hep3B cell viability, migration, and invasion, inactivated HIF-1α/HSP70 signaling, inhibited anti-apoptotic B-cell lymphoma (Bcl-2) activity, and increased the expression of pro-apoptotic Bcl-2-associated X protein (BAX) along with caspase-3. The synergistic effect was also confirmed using a 5-FU-resistant Hep3B mouse xenograft model in vivo. CONCLUSION: Nrf2 knockdown could improve the effect of As(2)O(3) on reversing drug resistance in 5-FU-resistant HCC cells.