Dihydromyricetin reverses MRP2-induced multidrug resistance by preventing NF-κB-Nrf2 signaling in colorectal cancer cell

Abstract

Backgroud: Dihydromyricetin (DMY), a natural flavonoid compound from the leaves of the Chinese medicinal herb Vitis heyneana, has been shown to have the potential to combat chemoresistance by inhibiting Nrf2/MRP2 signaling in colorectal cancer (CRC) cells. However, the precise underlying molecular mechanism and its therapeutic target are not well understood.

Purpose: Our study aims to investigate the effects of DMY on multidrug resistance (MDR), and elucidate the underlying mechanisms.

Study design: In vitro, HCT116/OXA and HCT8/VCR cells were employed as our MDR models. The cells were treated with DMY (50 µM) or MK-571 (50 µM) plus oxaliplatin (OXA) (10 µM) or vincristine (VCR) (10 µM) for 48 h. In vivo, we used BALB/c mice as a CRC xenograft mouse model. BALB/c mice were given DMY (100 mg/kg), OXA (5 mg/kg) and DMY (100 mg/kg) combined with OXA (5 mg/kg) via intraperitoneal route every 2 days per week for 4 weeks.

Methods: We used MTT and colony forming assays to detect DMY's ability to reverse MDR. Flow cytometric analysis was used to detect apoptosis. Immunocytochemistry was used to detect the localization of Nrf2 and NF-κB/p65. Western blot, qRT-PCR and reporter gene assays were employed to measure the protein and gene transcriptional levels (MRP2, Nrf2, NF-κB/p65). Moreover, chromatin immunoprecipitation (ChIP) assay was used to investigate the endogenous promoter occupancy of NF-κB/p65. Finally, immunohistochemistry and TUNEL staining were used to detect protein expression and apoptosis in vivo.

Results: DMY restored chemosensitivity (OXA and VCR) by inhibiting both MRP2 expression and its promoter activity in HCT116/OXA and HCT8/VCR cell lines. Furthermore, DMY could inhibit NF-κB/p65 expression, reducing NF-κB/p65 translocation to the nucleus to silence Nrf2 signaling, which is necessary for MRP2 expression. Overexpressing NF-κB/p65 expression reduced the reversal effect of DMY. In addition, NF-κB/p65 regulated Nrf2 expression by directly binding to its specific promoter region and activating its transcription. Finally, we proved that the combination of OXA and DMY has a synergistic tumor suppression effect in vivo.

Conclusion: Our study provided a novel mechanism of DMY boosted chemosensitivity in human CRC. The downstream signals of DMY, NF-κB or Nrf2 could also be potential targets for the treatment of CRC.

Keywords: Colorectal cancer; Dihydromyricetin; MRP2; Multidrug resistance; NF-κB.