SBF-1 downregulated the AR/IGF1 and its subsequent IGF-1/AKT/ FOXO1/PCNA signaling via directly binding to AR
Considering the important role of AR signaling in the growth of prostate cancer cells, we next examined the effect of SBF-1 on the signal of AR/IGF-1 and its subsequent IGF-1/AKT/FOXO1/PCNA. As shown in Fig. 2A, the expressions of IGF-1, PCNA, Bcl-2, pARS515, pAktS473 and pFOXO1S256 were greatly decreased in both LNCaP and PC3/AR+ cells by SBF-1 without affecting the total AR, AKT1, and FOXO1 expressions. This result suggests that SBF-1 downregulated AR/IGF-1 and IGF-1/AKT/FOXO1/PCNA pathways. It should be noted that the activation but not the expression of key enzymes or transcription factors was inhibited in the effect of SBF-1, which hints that the target of SBF-1 may be in the up-stream of AR signaling. To further confirm the effect of SBF-1 on the signaling, we used DHT to stimulate AR. As the result, DHT greatly increased the AR expression as well as the AR phosphorylation and the expressions of products IGF-1 and PCNA in both pathways. Against this, SBF-1 greatly blocked DHT-increased expressions of AR, pARS515, IGF-1, and PCNA in both ARWT and ARmutant cells (Fig. 2B). This result indicates dual effects of SBF-1 on AR/IGF1 axis and their down-stream signaling. Also, as seen in Fig. 2C, SBF-1 strongly suppressed the mRNA expression of IGF-1 and PCNA in either DHT presence or not. Especially in the case that DHT doubled the IGF-1 mRNA expression, SBF-1 still showed an almost complete inhibition. These findings suggest a possibility of SBF-1 to directly block the gene transcription mediated by AR.
To examine how SBF-1 affects AR and its subsequent signaling, we hypothesized that SBF-1 might directly bind to AR since it is a steroidal glycoside. We first conducted MST technique to examine whether there is any binding between SBF-1 and AR. The purified ARWT was tagged with GFP tag and transfected into HEK293T cells and the total cell lysis was extracted and incubated with different doses of SBF-1 to determine the binding affinity between SBF-1 and AR. As shown in Fig. 2D, the binding affinity between SBF-1 and AR is strong enough to conclude the binding, and the measured affinity was about 321 nM. Furthermore, we used ITC technique to confirm the binding between SBF-1 and AR. Namely, the purified ARWT was used along with SBF-1, and there is actually a strong binding with 2.95×10-5 M of the detected ∆K for the reaction between SBF-1 and ARWT protein (Fig. 2E).
Then, the polarity shift assay was performed to check whether the ligand-binding domain (AR-LBD) is the binding site of SBF-1. Flag-ARWT was transfected into HEK293T and then purified with the cleavage of Flag tag and incubated with fluorescent substrate along with the addition of SBF1 or DHT. When monitored as a fluorescent reading output, a significant shift in the polarization was detected to reveal a competitive replacement of substrate binding to AR-LBD by DHT, which confirmed the direct binding of DHT to the AR-LBD. However, there was no shifting signal in the case of SBF-1, which reveals that SBF-1 dose not bind to the AR-LBD at all (Fig. 2F). Above findings suggest that SBF-1 binds to the AR protein but AR-LBD is not the binding site.