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2-Methoxystypandrone inhibits signal transducer and activator of transcription 3 and nuclear factor-κB signaling by inhibiting Janus kinase 2 and IκB kinase.

Kuang S, Qi C, Liu J, Sun X, Zhang Q, Sima Z, Liu J, Li W, Yu Q - Cancer Sci. (2014)

Bottom Line: We have identified 2-methoxystypandrone (2-MS) from a traditional Chinese medicinal herb Polygonum cuspidatum as a novel dual inhibitor of JAK2 and IKK. 2-MS inhibits both interleukin-6-induced and constitutively-activated STAT3, as well as tumor necrosis factor-α-induced NF-κB activation. 2-MS specifically inhibits JAK and IKKβ kinase activities but has little effect on activities of other kinases tested.The inhibitory effects of 2-MS on STAT3 and NF-κB signaling can be eliminated by DTT or glutathione and can last for 4 h after a pulse treatment.We propose that the natural compound 2-MS, as a potent dual inhibitor of STAT3 and NF-κB pathways, is a promising anticancer drug candidate.

View Article: PubMed Central - PubMed

Affiliation: Division of Tumor Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.

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Related in: MedlinePlus

The covalent interaction of 2-methoxystypandrone (2-MS) with DTT. (a) The Michael addition reaction of α,β-unsaturated carbonyl of 2-MS with DTT; (b) TLC photography of 2-MS, DTT and Michael adducts of DTT to 2-MS under the ultraviolet light, 366 nm. TLC was performed on silica gel F254 TLC plates and was scanned using the Camag TLC visualizer. The No. III plate was developed in CHCl3-MeOH (4:1, v/v) solvent system, the spots in the No. III plate: 1: 2-MS (red); 2: 2-MS+DTT (yellow); 3: DTT. (c) Detection of a mixture of DTT, 2-MS and Michael adduct of DTT to 2-MS on the silica gel plate by a negative-mode electrospray ionization mass spectrometry (ESI-MS). The TLC bands of Michael adducts of DTT to 2-MS were scraped from the plates and eluted in acetonitrile. The resultant solution was filtered through a 45 μM filter and then collected for ESI-MS analysis.
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fig06: The covalent interaction of 2-methoxystypandrone (2-MS) with DTT. (a) The Michael addition reaction of α,β-unsaturated carbonyl of 2-MS with DTT; (b) TLC photography of 2-MS, DTT and Michael adducts of DTT to 2-MS under the ultraviolet light, 366 nm. TLC was performed on silica gel F254 TLC plates and was scanned using the Camag TLC visualizer. The No. III plate was developed in CHCl3-MeOH (4:1, v/v) solvent system, the spots in the No. III plate: 1: 2-MS (red); 2: 2-MS+DTT (yellow); 3: DTT. (c) Detection of a mixture of DTT, 2-MS and Michael adduct of DTT to 2-MS on the silica gel plate by a negative-mode electrospray ionization mass spectrometry (ESI-MS). The TLC bands of Michael adducts of DTT to 2-MS were scraped from the plates and eluted in acetonitrile. The resultant solution was filtered through a 45 μM filter and then collected for ESI-MS analysis.

Mentions: As shown in Fig. 1, 2-MS contains three α,β-unsaturated carbonyl groups which are reactive with thiols. The thiols of cysteines in JAK2 and IKKβ are reported to be important for their kinase activities and several compounds with thiol-reactive groups have been found to covalently bind to Cysteine 179 of IKKβ and inhibit its kinase activity.(23–26) Therefore, we investigated whether the inhibitory effects of 2-MS on JAK2 or IKKβ kinase activity were through interacting with their cysteine thiols. We used two thiol-containing compounds, DTT or GSH, to compete with JAK2 or IKKβ on interacting with 2-MS to test whether the inhibitory effects of 2-MS on the STAT3 or NF-κB pathway could be alleviated by the two thiol-containing compounds. As shown in Figure. 5(a) and (c), after incubation with DTT or GSH, the inhibitory effects of 2-MS on STAT3 and NF-κB pathways were abolished. Therefore, 2-MS may inhibit the STAT3 and NF-κB pathways through interacting with the thiols of JAK2 and IKKβ via its α,β-unsaturated carbonyl groups. To further demonstrate the 2-MS-thiol interaction, 2-MS was mixed with DTT to induce a chemical reaction of Michael addiction. The proposed reaction site of 2-MS is illustrated in Fig. 6(a). The Michael adducts of DTT to 2-MS were then examined using TLC and ESI-MS analysis, which detected one product at m/z 412.97 [2-MS + DTT-H]−, indicating the addition of DTT to 2-MS (Fig. 6b,c). The results strongly support the possibility that 2-MS reacts with the cysteine thiols of JAK2 and IKKβ.


2-Methoxystypandrone inhibits signal transducer and activator of transcription 3 and nuclear factor-κB signaling by inhibiting Janus kinase 2 and IκB kinase.

Kuang S, Qi C, Liu J, Sun X, Zhang Q, Sima Z, Liu J, Li W, Yu Q - Cancer Sci. (2014)

The covalent interaction of 2-methoxystypandrone (2-MS) with DTT. (a) The Michael addition reaction of α,β-unsaturated carbonyl of 2-MS with DTT; (b) TLC photography of 2-MS, DTT and Michael adducts of DTT to 2-MS under the ultraviolet light, 366 nm. TLC was performed on silica gel F254 TLC plates and was scanned using the Camag TLC visualizer. The No. III plate was developed in CHCl3-MeOH (4:1, v/v) solvent system, the spots in the No. III plate: 1: 2-MS (red); 2: 2-MS+DTT (yellow); 3: DTT. (c) Detection of a mixture of DTT, 2-MS and Michael adduct of DTT to 2-MS on the silica gel plate by a negative-mode electrospray ionization mass spectrometry (ESI-MS). The TLC bands of Michael adducts of DTT to 2-MS were scraped from the plates and eluted in acetonitrile. The resultant solution was filtered through a 45 μM filter and then collected for ESI-MS analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317813&req=5

fig06: The covalent interaction of 2-methoxystypandrone (2-MS) with DTT. (a) The Michael addition reaction of α,β-unsaturated carbonyl of 2-MS with DTT; (b) TLC photography of 2-MS, DTT and Michael adducts of DTT to 2-MS under the ultraviolet light, 366 nm. TLC was performed on silica gel F254 TLC plates and was scanned using the Camag TLC visualizer. The No. III plate was developed in CHCl3-MeOH (4:1, v/v) solvent system, the spots in the No. III plate: 1: 2-MS (red); 2: 2-MS+DTT (yellow); 3: DTT. (c) Detection of a mixture of DTT, 2-MS and Michael adduct of DTT to 2-MS on the silica gel plate by a negative-mode electrospray ionization mass spectrometry (ESI-MS). The TLC bands of Michael adducts of DTT to 2-MS were scraped from the plates and eluted in acetonitrile. The resultant solution was filtered through a 45 μM filter and then collected for ESI-MS analysis.
Mentions: As shown in Fig. 1, 2-MS contains three α,β-unsaturated carbonyl groups which are reactive with thiols. The thiols of cysteines in JAK2 and IKKβ are reported to be important for their kinase activities and several compounds with thiol-reactive groups have been found to covalently bind to Cysteine 179 of IKKβ and inhibit its kinase activity.(23–26) Therefore, we investigated whether the inhibitory effects of 2-MS on JAK2 or IKKβ kinase activity were through interacting with their cysteine thiols. We used two thiol-containing compounds, DTT or GSH, to compete with JAK2 or IKKβ on interacting with 2-MS to test whether the inhibitory effects of 2-MS on the STAT3 or NF-κB pathway could be alleviated by the two thiol-containing compounds. As shown in Figure. 5(a) and (c), after incubation with DTT or GSH, the inhibitory effects of 2-MS on STAT3 and NF-κB pathways were abolished. Therefore, 2-MS may inhibit the STAT3 and NF-κB pathways through interacting with the thiols of JAK2 and IKKβ via its α,β-unsaturated carbonyl groups. To further demonstrate the 2-MS-thiol interaction, 2-MS was mixed with DTT to induce a chemical reaction of Michael addiction. The proposed reaction site of 2-MS is illustrated in Fig. 6(a). The Michael adducts of DTT to 2-MS were then examined using TLC and ESI-MS analysis, which detected one product at m/z 412.97 [2-MS + DTT-H]−, indicating the addition of DTT to 2-MS (Fig. 6b,c). The results strongly support the possibility that 2-MS reacts with the cysteine thiols of JAK2 and IKKβ.

Bottom Line: We have identified 2-methoxystypandrone (2-MS) from a traditional Chinese medicinal herb Polygonum cuspidatum as a novel dual inhibitor of JAK2 and IKK. 2-MS inhibits both interleukin-6-induced and constitutively-activated STAT3, as well as tumor necrosis factor-α-induced NF-κB activation. 2-MS specifically inhibits JAK and IKKβ kinase activities but has little effect on activities of other kinases tested.The inhibitory effects of 2-MS on STAT3 and NF-κB signaling can be eliminated by DTT or glutathione and can last for 4 h after a pulse treatment.We propose that the natural compound 2-MS, as a potent dual inhibitor of STAT3 and NF-κB pathways, is a promising anticancer drug candidate.

View Article: PubMed Central - PubMed

Affiliation: Division of Tumor Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.

Show MeSH
Related in: MedlinePlus