Inhibition of the Glutaredoxin System Increases Doxorubicin Sensitivity in Hepatocellular Carcinoma By Impairing the Nrf2-Dependent Antioxidant Response
MetadataShow full item record
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 apoptosis and doxorubicin sensitivity in HepG2 by impairing the Nrf2-dependent antioxidant response. Methods: Hepatocellular carcinoma tissue and neighboring healthy liver tissue was obtained from five patients from Sun-Yat-Sen Hospital and tested for Grx1 and Grx2 expression levels using western blot. HepG2 cells were transfected with Grxs or scramble shRNA vector. Scramble sh-RNA, Grx1 shRNA, and Grx2shRNA HepG2 cells were treated with 1 uM and 10 uM doxorubicin, and 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) in whole cell lysates and mitochondrial fractions were measured by immunoblotting using anti-PSSG antibody. Western blot was used to also examine the expression levels of Nrf2 and its downstream proteins in Grxs-inhibited cells before and after doxorubicin treatment. Nrf2 translocation assay and co-immunoprecipitation with Grxs and PSSG was also performed. Immunostaining was done to confirm Grx2’s presence in the nucleus and Grx2-Nrf2 binding. NucBlue live cell staining was performed to analyze Grx2’s possible function in the nucleus. Antioxidant gene screening for 48 Nrf2-pathway related genes for scramble and Grxs shRNA after doxorubicin treatment was analyzed. Results: All five patients in our clinical study showed much higher Grx1 and Grx2 levels in cancerous tissue than in normal liver tissue. shRNA transfection gave a 50-70% Grxs knockdown. Grxs inhibition caused increased doxorubicin sensitivity with approximately a 20% lower cell viability at 1 and 10 uM doxorubicin treatment, higher pro-apoptotic protein expression levels, and increased glutathionylation than control. Moreover, actin glutathionylation increased in Grxs shRNA cells. Grxs inhibition also decreased the expression of antioxidant enzyme transcription factor regulator, Nrf2, and its downstream antioxidant proteins like HO-1, catalase, thioredoxin, and NQO1 especially after doxorubicin treatment. Nrf2’s presence in the nucleus and cytoplasm decreased with glutaredoxin knockdown. Doxorubicin treatment enhanced Grx1 and Grx2 binding with Nrf2 in scramble shRNA HepG2 cells. Glutaredoxin inhibition also significantly increased Nrf2 glutathionylation, indicating lower Nrf2 activation. Immunostaining showed abundant amount of Grx2 in the nucleus and that Grx2 specifically interacts and repairs Nrf2 in the nucleus. Grx2 may also be involved in DNA repair and synthesis in the nucleus. Gene screening also showed significant decrease in mRNA levels of Bcl2, GSTs, and Prdx6 with Grxs inhibition after doxorubicin treatment. Conclusions: Grx1 is vital to repair Nrf2 in the cytoplasm, whereas Grx2 repairs Nrf2 in the nucleus, and if inhibited, can cause an increase in Nrf2 glutathionylation and inactivation. Grxs inhibition causes increased doxorubicin sensitivity and apoptosis of hepatocellular carcinoma by attenuating Nrf2 and its downstream antioxidant genes’ activation, making it an ideal pharmacological target for future anti-liver cancer treatment.