Abstract: Background and purpose: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively kill tumor cells, but many tumors are resistant to it. The aim of our study was to investigate the effects of gambognic acid (GA) combining TRAIL on the growth of subcutaneous tumor xenografts in nude mice established from human colon cancer cell line HT-29, and to explore the mechanisms related to the effects of GA combined with TRAIL on apoptosis of human colon cancer HT-29 cells. Methods: A nude mouse model of colon cancer was established by subcutaneous inoculation of human colon cancer cell line HT-29. The effects of TRAIL or/and GA on transplanted tumor in nude mice were measured. The histopathological changes of tumor tissues were observed by H-E staining, and the tumor cell apoptosis was detected by TUNEL assay. Transfection of HT-29 cells with the siRNA caused a significant reduction in Nrf2 protein expression. Then Annexin Ⅴ-FITC apoptosis kit was used to detect the cell apoptosis, and the generation of reactive oxygen species (ROS) was assayed using flow cytometry. The mRNA expressions of Nrf2, Bcl-2, Bax and DR5 were determined by RT-PCR. Results: TRAIL combined with GA significantly promoted the inhibitory effect of TRAIL on the growth of transplanted tumor in nude mice (the inhibition rate reached 67.0%), increased the apoptosis of HT-29 cells, promoted the decrease in the expression of Nrf2 and Bcl-2, and potentiated the expression of Bax and DR5. Compared with control siRNA, Nrf2 interference markedly increased the apoptosis of HT-29 cells induced by TRAIL, enhanced the ROS level, down-regulated the expression of Nrf2 and Bcl-2, and up-regulated the expression of Bax and DR5. Conclusion: GA reverses the TRAIL resistance in HT-29 cells in vivo and in vitro by upregulating Nrf2 and promoting ROS-activated mitochondrial apoptosis pathway and death receptor pathway.

Key words: Gambognic acid, Tumor necrosis factor-related apoptosis-inducing ligand, HT-29 cel1s, Nrf2, Bcl-2, Bax, DR5