br AFL reduces Twist stability br
3.4 AFL reduces Twist1 stability
Since Twist1 has been identified as the target of AFL, we further determined if AFL affected the stability of Twist1. A549 Conessine were subject to AFL treatment and the level of Twist1 was examined. We found that Twist1 levels were reduced by AFL administration (Figure 4A). Furthermore, the decrease in Twist1 protein abundance was also dependent upon AFL dosage (Figure 4A). Notably, the effect of AFL on Twist1 protein expression was not evident at transcription level (Figure 4B). We also dynamically measured the Twist1 variation after AFL treatment and the results confirmed that AFL could decrease the stability of Twist1 (Figure 4C). The half- life of Twist1 was shifted from 9.167 h (95% confidence interval [8.099, 10.559]) to 3.061 h (95% confidence interval [2.498, 3.950]) after AFL treatment (Figure 4D). As expected, we found that AFL treatment substantially increased the ubiquitination of Twist1 (Figure 4E). These results suggested that AFL could promote Twist1 turnover by enhancing Twist1 ubiquitination.
3.5 Twist1 mediated the effect of AFL
Since AFL could target Twist1, we next checked whether Twist1 indeed affected AFL- mediated tumor suppression. As demonstrated above, AFL could inhibit migration of NSCLC cells (Figures 5A and 5B). However, re- introducing Twist1 into A549 cells could partially reverse the effect of AFL (Figures 5A and 5B). AFL treatment reduced Twist1 abundance and simultaneously increased E-cadherin and reduced N-cadherin and vimentin expression (Figure 5C). Consistently, overexpression of Twist1 with concomitant AFL treatment partially rescued the expression of EMT markers (Figure 5C). Twist1 overexpression indeed significantly elevated endogenous Twist1 protein levels (Figure 5D). In vivo experiments further showed that AFL treatment repressed the metastasis of NSCLC cells (Figures 5E and 5F). However, Twist1 overexpression could compensate for the inhibitory effect of AFL treatment and ‘reactivate’ the metastatic potential of NSCLC cells (Figures 5E and 5F). These data again consolidated that the effect of AFL could be mediated by Twist1.
3.6 AFL reverses cisplatin resistance in NSCLC cells
Previous report argues that Twist1 overexpression contributes to cisplatin resistance in NSCLC (e.g. A549 and H1299 cell lines) . Since AFL could lower the Twist1 protein stability, we then asked whether AFL may sensitize NSCLC cells to cisplatin treatment. Consistent with previous work, treatment with cisplatin in A549 and H1299 cells could only slightly lower the viability of NSCLC cells (Figure 6A). However, AFL treatment could sensitize A549 and H1299 cells to cisplatin (Figure 6A). We further knockdown Twist1 expression (Figure 6B) and found that lowering Twist1 could also increase the responsiveness to cisplatin in NSCLC cells (Figure 6C). As expected, co-treatment of NSCLC cells with AFL and cisplatin substantially increased the apoptosis whereas single cisplatin treatment only induced minimal cleaved PARP and caspase-3 (Figure 6D). The metastatic ability of NSCLC cells was
markedly lowered when AFL and cisplatin were co-treated (Figures 6E and 6F). By Ki-67 staining, we found AFL could also reverse cisplatin resistance and significantly decrease proliferation of NSCLC cells (Figures 6G and 6H, P < 0.01). These data suggested that AFL may sensitize NSCLC cells to cisplatin treatment in cisplatin resistant cells.
A new natural product, atalantraflavone (AFL), has been identified from the leaves of Atalantia monophylla (L.) DC . However, the function of atalantraflavone in lung cancer remains totally unknown. In present study, we for the first time showed that atalantraflavone may play a tumor suppressive role in NSCLC. Mechanistic studies revealed that atalantraflavone could target Twist1 and lower its stability. Meanwhile, both in vivo and in vitro experiments supported that atalantraflavone may also sensitize NSCLC cells to cisplatin in cisplatin resistant cells. Therefore, our current study has provided a possibility of atalantraflavone treatment during therapeutic intervention for NSCLC.