Supplementary Material for: Anti-Cancer Effects of Radix Angelica Sinensis (Danggui) and N-Butylidenephthalide on Gastric Cancer: Implications for REDD1 Activation and mTOR Inhibition

Background/Aims: Radix Angelica Sinensis (danggui in Chinese) is widely used in traditional chinese medicine (TCM). N-butylidenephthalide (BP), a bioactive compound in danggui, is a potential antitumor agent for various cancer types. However, its clinical effect and mechanism in the treatment of gastric cancer remain undetermined. Methods: The in vivo protective effect of danggui in patients with gastric cancer were validated using data from Taiwan’s National Health Insurance Research Database (NHIRD). The genes induced by BP-treatment were analyzed by whole transcriptome RNA sequencing (RNA-seq) and validated by real-time PCR, western blot and siRNA transfection. The effect of BP on AGS cell migration and invasion was evaluated in transwell assays. The antitumor effects of BP were evaluated in vivo in an AGS xenograft animal model. Results: Danggui users were found to have an increased survival rate when compared with danggui nonusers (log-rank test p = 0.002) . The use of danggui highly associated with decreased mortality (the adjusted hazard ratio (HR) of danggui user was 0.72 [95 % CI, 0.57-0.92] (p = 0.009). The in vitro results showed that BP inhibited gastric cancer cell proliferation, and triggered cellular apoptosis depending on the activation of mitochondrial apoptotic pathway. Using RNA-seq analysis we found that REDD1 was the highest transcript induced by BP in gastric cancer cells. BP induce an increase of REDD1 expression that inhibits mTOR signaling, thus inhibiting gastric cancer growth. We used RNA interference to demonstrate that the knock-down of REDD1 attenuated the BP-induced mTORC1 activation and growth inhibition. BP suppressed the growth of AGS xenografts tumor in vivo. Conclusion: Danggui can prolong the survival rate of gastric cancer patients in Taiwan. BP caused gastric cancer cell death through the activation of mitochondria-intrinsic pathway and induced the REDD1 expression leading to mTOR signal pathway inhibition in gastric cancer cells. BP inhibited the in vivo growth of AGS xenograft tumors. These results may provide the basis for a new therapeutic approach toward the treatment of gastric cancer progression.