Eriocalyxin B Inhibits STAT3 Signaling by Covalently Targeting STAT3 and Blocking Phosphorylation and Activation of STAT3

<div><p>Activated STAT3 plays an important role in oncogenesis by stimulating cell proliferation and resisting apoptosis. STAT3 therefore is an attractive target for cancer therapy. We have screened a traditional Chinese herb medicine compound library and found Eriocalyxin B (EB), a diterpenoid from Isodon eriocalyx, as a specific inhibitor of STAT3. EB selectively inhibited constitutive as well as IL-6-induced phosphorylation of STAT3 and induced apoptosis of STAT3-dependent tumor cells. EB did not affect the upstream protein tyrosine kinases or the phosphatase (PTPase) of STAT3, but rather interacted directly with STAT3. The effects of EB could be abolished by DTT or GSH, suggesting a thiol-mediated covalent linkage between EB and STAT3. Site mutagenesis of cysteine in and near the SH2 domain of STAT3 identified Cys712 to be the critical amino acid for the EB-induced inactivation of STAT3. Furthermore, LC/MS/MS analyses demonstrated that an α, β-unsaturated carbonyl of EB covalently interacted with the Cys712 of STAT3. Computational modeling analyses also supported a direct interaction between EB and the Cys712 of STAT3. These data strongly suggest that EB directly targets STAT3 through a covalent linkage to inhibit the phosphorylation and activation of STAT3 and induces apoptosis of STAT3-dependent tumor cells.</p></div

DTT and GSH abolished the EB-caused JAK-STAT3 inhibition.

<p><b>(A)</b> A549 cells were pretreated with DTT (0.5 mM), GSH (2 mM), EB (20 μM) or their mixture (EB and DTT/GSH were pre-incubated at RT for 30 min) for 2 h and then stimulated with IL-6 for 15 min. Whole cell lysates were processed for western blot analysis using antibodies as indicated. <b>(B)</b> A549 cells were cultured in the presence of indicated concentrations of EB or EB + DTT (0.5 mM) for 48 h and then the viability of A549 cells was analyzed by MTT assay. <b>(C)</b> EB (50 μM) was incubated with GSH (250 μM) at 37°C for 2 h, and the products were resolved by mass spectrometry. A possible Michael addition reaction between EB and GSH is illustrated. The black arrows indicated the molecular weights of EB+GSH and EB+2GSH.</p

EB covalently targeted STAT3 Cys712.

<p><b>(A)</b> A549 cells were transfected with plasmids encoding the myc-tagged wild-type, C418S, C426S, C468S, C542S, C550S, C687S, C712S, or C718S mutation of STAT3 for 24 h. The cells were then treated with EB at indicated concentrations for 2 h before stimulation with IL-6 for 15 min. Whole cell lysates were processed for western blot analysis with anti-STAT3 or anti-p-STAT3 Tyr705 antibodies. The molecular weight of the exogenous myc-tagged STAT3 is 95 kDa, while the endogenous STAT3 is 87 kDa. <b>(B)</b> The sequence comparison of all human STAT family members showing that the cysteine at position 712 of STAT3 is unique (highlighted in gray). <b>(C)</b> EB (10 mM, 3.75 μL) was incubated with the STAT3 Cys712-containing peptide FT-8 (FICVTPTT) (500 μM, 75 μL) at 37°C for 2 h, and the products were resolved by mass spectrometry. The molecular weight of peptide FT-8 is 881.3 and the molecular weight of the covalent product between EB and FT-8 is 1225.5. <b>(D)</b> EB (10 mM, 3.75 μL) was incubated with the peptide FT-8 (FICVTPTT) (500 μM, 75 μL) at 37°C for 2 h, and the products were analyzed by MS/MS. b3, b4, b5, b6, b7 represent the fragmented EB-containing peptides. C* represents the Cys bound by EB. <b>(E)</b> EB (20 mM, 3.75 μL) was incubated with peptide QP-10 (QFTKCCPPKP) (500 μM, 75 μL) at 37°C for 2 h, and the products were analyzed by mass spectrometry. <b>(F)</b> Computational modeling of the interaction between EB and STAT3. The crystal structure of STAT3 was obtained from PDB (Protein Data Bank). Oxygen atoms of EB were shown in red. Hydrogen bonds between Gln644, Asn647, and EB were shown in purple. The predicted distance between the α, β-unsaturated carbonyl and the thiol of Cys712 is 4.3 À as indicated.</p

EB specifically inhibited the activation of STAT3.

<p><b>(A)</b> Chemical structure of EB. <b>(B)</b> HepG2/STAT3-luceferase reporter cells were pretreated with EB at indicated concentrations for 2 h, and luciferase activity was measured following stimulation with IL-6 (50 ng/ml) for 5 h. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001 versus the control group without EB but with IL-6 stimulation. <b>(C)</b> A549 cells were pretreated with EB at indicated concentrations for 2 h before stimulation by IL-6 (10 ng/ml) for 15 min. Whole cell lysates were processed for western blot analysis with the indicated antibodies. <b>(D)</b> A549 cells were pretreated with 20 μM EB for various time periods (0–120 min) before stimulation by IL-6 for 15 min. Whole cell lysates were processed for western blot analysis with the indicated antibodies. <b>(E)</b> MDA-MB-231 and MDA-MB-468 cells were treated with EB at indicated concentrations for 2 h. Whole cell lysates were processed for western blot analysis with indicated antibodies. <b>(F)</b> A549 cells were cultured on coverslips in serum-free medium for 24 h. The cells were then pretreated with vehicle or 10 μM EB for 2 h, followed by 30 min stimulation with IL-6. The cells on the coverslips were then processed for immunochemical staining with an anti-STAT3 antibody or nuclei staining with Diamidino-phenyl-indole. (<b>G</b>) EB and nuclear extract from A549 cells that contained activated STAT3 proteins were preincubated for 1h prior to addition of DNA probe. The STAT3 DNA-binding activity was assessed by EMSA. The oligo band shift caused by STAT3 binding and the super shift caused by the anti-STAT3 antibody binding are indicated. <b>(H)</b> A549 cells were treated with EB at indicated concentrations for 2 h, followed by stimulation with IFN-γ for 15 min. Whole cell lysates were processed for western blot analysis using the antibodies as indicated.</p

EB did not inhibit the upstream components of STAT3 signaling.

<p><b>(A)</b> IL-6 (10 ng/ml) was pretreated with EB at indicated concentrations in vitro for 30 min, and then was used to stimulate A549 cells for 15 min. Whole cell lysates were processed for western blot analysis with the indicated antibodies. <b>(B)</b> A549 cells were serum starved for 24 h, and then treated with EB at indicated concentrations for 2 h before stimulation with IL-6 for 15 min. Whole cell lysates were analyzed by western blot using the indicated antibodies. <b>(C)</b> A549 cells were pretreated with indicated concentrations of EB for 2 h, whole cell lysates were processed for western blot analysis using antibodies as indicated.</p

EB inhibited tumor cell growth by inducing apoptosis.

<p><b>(A)</b> MDA-MB-468, A549, MDA-MB-453 cells (3.0*10⁵ per well) were treated with EB or Stattic at indicated concentrations for 2 h. Whole cell lysates were processed for western blot analyses using antibodies as indicated. <b>(B)</b> MDA-MB-468, A549, MDA-MB-453 cells were treated with EB or Stattic at indicated concentrations for 48 h. The viability of the three cell lines was analyzed by MTT assay. <b>(C)</b> A549 cells were treated with EB at indicated concentrations for 12 h. The whole cell lysates were processed for western blot analysis using antibody as indicated. <b>(D)</b> A549 cells were treated with EB (20 μM) for various time periods (0–48 h). Whole cell lysates were processed for western blot analysis using antibody as indicated.</p

Effects of EB (50 μM) on in vitro kinase activities.

<p>The kinase assay was performed as described in the Methods.</p><p>Effects of EB (50 μM) on in vitro kinase activities.</p

EB did not enhance the activity of tyrosine phosphatases.

<p><b>(A)</b> A549 cells were stimulated by IL-6 (10 ng/ml) for 15 min. The media were then replaced by fresh media without IL-6 and incubated with or without EB (20 μM) for different time periods (0–240 min). Whole cell lysates were processed for western blot analysis using antibodies as indicated. <b>(B)</b> A549 cells were pre-treated with sodium orthovanadate (50 μM), EB (20 μM) or their mixture for 2 h, then stimulated with IL-6 for 15 min. Whole cell lysates were processed for western blot analysis using antibodies as indicated.</p

EB and IFN-γ synergistically inhibited tumor cell growth.

<p>(<b>A</b>) A549 cells were plated into a Real-Time Cell Analyzer (RTCA) for 24 h. IFN-γ (10000 U), EB (1 μM), or their mixture were then added. The cell growth was recorded by the Real-Time Cell Analyzer.</p

Flow chart of the iterative Bayesian estimation.

<p>Flow chart of the iterative Bayesian estimation.</p