P-0079 Nrf2 Knockdown by Sirna Enhance the Sensitivity to Chemotherapy in Hepatocellular Carcinoma

Abstract

IntroductionNF-E2-related factor 2 (Nrf2), acts as a key transcription regulator in cytoprotection, is essential for the malignant biological behavior of cancer including proliferation, invasion, metastasis, and drug resistance. It has been reported that Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs in lung cancer, esophageal squamous cancer and breast cancer cells. In this study, we investigated the role of Nrf2 in terms of Hepatocellular Carcinoma (HCC) cells proliferation and drug resistance. MethodsMTT colorimetric assay and flow cytometry were performed to investigate the sensitivity of different HCC cells including HepG2, Hep3B and SMCC-7721 to Cisplatin and Fluorouracil. Immunofluorescent assay was applied to detect the relationship between Nrf2-subcellular location and the chemotherapy sensitivity of HCC cells. Plasmids containing Nrf2-small interfering RNA (siRNA) or non-targeting vector control siRNA were transfected into HCC cells. The expression of Nrf2 was measured in HCC cells stably expressing Nrf2-siRNA by Western blots and RT-PCR assays, and the subcellular localization of Nrf2 was analyzed by immunofluorescent assay. To evaluate how the Nrf2 knockdown affected susceptibility to chemotherapeutic drugs, MTT and flow cytometry were done again in vitro. ResultsThe susceptibility to chemotherapeutic drugs difference of all crowds was ordered from sensitivity to resistant as HepG2, Hep3B and SMCC-7721. Immunofluorescent assay showed that the distribution of Nrf2 in HCC cells markedly decreased in the cytoplasm and relatively increased in the nucleus with the decreased sensitivity. However, combination of Cisplatin or Fluorouracil and knock-out Nrf2 by siRNA significantly suppressed cell proliferation and increased cell apoptosis, especially in drug-resistant cells SMCC-7721. In SMCC-7721 cells, Nrf2-siRNA suppressed the nuclear transfer of Nrf2 induced by chemotherapy drugs resulted in enhancement of chemotherapy sensitivity. ConclusionOur findings provide evidences that Nrf2-siRNA acts as a chemotherapy-sensitizing agent to enhance the efficacy of chemotherapy and thus can be further used as an adjuvant treatment in chemotherapy of hepatocellular carcinoma.