Inhibition of the Glutaredoxin System Increases Doxorubicin Sensitivity in Hepatocellular Carcinoma by Impairing the Nrf2-dependent Antioxidant Response




Xavier, Christy
Wu, Hongli
Liu, Xiaobin
Nguyen, Benjamin


Journal Title

Journal ISSN

Volume Title



Purpose: Hepatocellular carcinoma (HepG2) is the most common type of liver cancer, causing approximately 1.25 million deaths annually. Even with premier anti-cancer drugs like doxorubicin, the lethality of hepatocellular carcinoma has increased and is mainly attributed to growing drug resistance. Specifically, overexpression of key antioxidant enzymes such as the glutaredoxin system (Grxs) may enable drug resistance. Glutaredoxin is a powerful protective thiol repair enzyme that increases cancer cell survival. In this study, we explored a new anti-cancer strategy, the inhibition of Grxs, as a way to both increase doxorubicin sensitivity and reverse resistance in HepG2 by impairing the Nrf2-dependent antioxidant response. Methods: HepG2 cells (Sigma) were transfected with Grxs or scramble shRNA vector. HepG2 was treated with doxorubicin in a dose and time-dependent manner. Cell viability was measured by the WST-8 colorimetric assay. Western blot was performed to test expression levels of pro- and anti-apoptotic proteins like Bax, Bcl2, and cleaved caspase-3. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Western blot was used to also examine the expression levels of Nrf2 and its downstream genes in Grxs-inhibited cells before and after doxorubicin treatment. Nrf2 translocation assay and co-immunoprecipitation with Grxs and PSSG was also performed. Antioxidant gene screening for 91 Nrf2-pathway related genes for scramble and Grxs shRNA after doxorubicin treatment was analyzed. Results: shRNA transfection gave a 50-70% Grxs knockdown. Grxs inhibition caused increased doxorubicin sensitivity with lower cell viabilities, higher pro-apoptotic protein expression levels, and increased glutathionylation than control. Grxs inhibition also decreased the expression of antioxidant enzyme transcription factor regulator, Nrf2, and its downstream antioxidant genes like HO-1, catalase, thioredoxin, and NQO1 especially after doxorubicin treatment. Nrf2’s presence in the nucleus and cytoplasm decreased with glutaredoxin knockdown. Glutaredoxin inhibition also significantly increased Nrf2 glutathionylation. Gene screening also showed significant decrease in mRNA levels of Nrf2-pathway related genes with Grxs inhibition after doxorubicin treatment. Conclusions: Grxs inhibition causes increased doxorubicin sensitivity and apoptosis of hepatocellular carcinoma by attenuating Nrf2 and its downstream antioxidant genes activation.


Research Appreciation Day Award Winner - 2015 UNT System College of Pharmacy - Pharmacy Student Research Award