3.1 TA blocks the STAT3 signaling pathway
To identify natural compounds that inhibit the transcriptional activity
of STAT3, we used a luciferase reporter assay to screen natural
compounds as potential STAT3 pathway inhibitors. 293T cells were
stimulated with IL-6 to activate the STAT3 signaling pathway. After
incubation with these natural compounds for 12 h, the transfected cells
were collected and STAT3 transcriptional activity was detected by dual
luciferase assay. We identified a natural product, Trienomycin A (TA),
that potently reduced the transcriptional activity of STAT3. As shown in
Figure 1b, TA dose-dependently inhibited the IL-6-induced
transcriptional activity of STAT3.
As mentioned above, STAT3 has been considered as a potential anticancer
target. Accordingly, we next investigated whether TA interacted with
STAT3. We performed the molecular docking and binding free energy
(ΔGbind) calculation using the X-ray crystal structure
of the STAT3β/DNA complex (PDB:1BG1), and identified a potential binding
site on the surface of STAT3 that could reasonably accommodate TA
binding. As shown in Figure 1c, the oxygen atom of phenolic hydroxyl
group on C-22 of TA forms hydrogen bond with the hydrogen atom of Asn485
side-chain amide. The oxygen atom of methoxy group on C-3 of TA forms
hydrogen bond with the hydrogen atom of Lys488 side-chain amine. The
substituent group on C-11 of TA is very important for the combination
with STAT3, and the two oxygen atoms of its ester group form hydrogen
bonds with the hydrogen atoms of Lys244 side-chain amine, and the oxygen
atom (carbonyl oxygen atom on C-30) of its amide group forms hydrogen
bond with the hydrogen atom of His457 framework amide. Moreover, we
found that TA could fit to the hot spots of STAT3 protein with a binding
energy of -9.32 kcal/mol. Probably, TA directly interacted with STAT3,
inducing a change in the conformation of the protein, resulting in the
inhibitory effects on the STAT3 signaling pathway.
To further validate the direct interaction of TA with STAT3, the binding
affinity of TA with STAT3 was detected by the SPR assay. As shown in
Figure 1d, we observed that TA interacted with STAT3 protein with an
equilibrium dissociation constant (KD) of 18.00 µmol/L. In addition, the
structural analogues of TA could also bind directly to STAT3 with KD
values range from 4.42 µmol/L to 17.78 µmol/L (Figure S1a). As shown in
Figure S1b, the STAT3 inhibitors, C188-9 and BP-1-102, bound to STAT3
with a KD value of 3.71 µmol/L and 0.84 µmol/L, which were approximately
equal to values reported in literature, respectively (Bharadwaj et al.,
2016; Zhang et al., 2012). To sum up, all these data indicated that TA
most probably inhibited STAT3 signaling pathway by targeting STAT3.
We next examined whether TA could suppress the STAT3 pathway in
pancreatic cancer. Firstly, we searched the expression of p-STAT3
(Tyr705) protein in various cancer cell lines by using the Cancer Cell
Line Encyclopedia (CCLE). It was intriguing to find that p-STAT3
(Tyr705) protein was highly expressed in pancreatic cancer compared with
most other cancer cell lines such as gastric cancer, breast cancer,
ovarian cancer and so on (Figure S1c). In order to further detect the
expression and activation of STAT3, the expression levels of total STAT3
and p-STAT3 (Tyr705 and Ser727) were evaluated in human pancreatic
cancer cell lines (PANC-1, BxPC-3, MIA PaCa-2, Capan-2, CFPAC-1, AsPC-1,
SW1990, and HPAC). As demonstrated in Figure 1e, the PANC-1, CFPAC-1,
Capan-2, BxPC-3, and HPAC cell lines showed higher levels of p-STAT3
(Tyr705) protein compared with the rest. Based on these results, the
PANC-1, CFPAC-1, Capan-2, and BxPC-3 cell lines were selected for
subsequent experiments.
Subsequently, we investigated the effect of TA on the expression levels
of total STAT3, p-STAT3 (Tyr705 and Ser727), and STAT3-regulated
proteins in human pancreatic cancer cell lines. As shown in Figures 1f
and S1d, TA remarkably inhibited p-STAT3 (Tyr705) expression levels in a
dose-dependent manner, while had no concentration-dependent suppressive
effect on p-STAT3 (Ser727) and total STAT3 expression levels. TA also
significantly down-regulated the protein expression of STAT3 downstream
target genes (Cyclin D1, Survivin, and ICAM-1) in a dose-dependent way.
Taken together, these results demonstrated that TA substantially
inhibited the STAT3 signaling pathway.