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Gossypol induces apoptosis in multiple myeloma cells by inhibition of interleukin-6 signaling and Bcl-2/Mcl-1 pathway.

Sadahira K, Sagawa M, Nakazato T, Uchida H, Ikeda Y, Okamoto S, Nakajima H, Kizaki M - Int. J. Oncol. (2014)

Bottom Line: In addition, Mcl-1 was downregulated by Gos.Moreover, JAK2 inhibition mimicked the effect of Gos in OPM2 cells including Bcl-2 dephosphorylation and Mcl-1 downregulation.These results demonstrated that Gos induces apoptosis in MM cells not only through displacing BH3-only proteins from Bcl-2, but also through inhibiting IL-6 signaling, which leads to Bcl-2 dephosphorylation and Mcl-1 downregulation.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Department of Internal Medicine, Keio University School of Medicine, Tokyo 160-8582, Japan.

ABSTRACT
Multiple myeloma (MM) is a clonal plasma cell disorder affecting the immune system with various systemic symptoms. MM remains incurable even with high dose chemotherapy using conventional drugs, thus necessitating development of novel therapeutic strategies. Gossypol (Gos) is a natural polyphenolic compound extracted from cotton plants, and has been shown to possess anti-neoplastic activity against various tumors. Recent studies have shown that Gos is an inhibitor for Bcl-2 or Bcl-XL acting as BH3 mimetics that interfere interaction between pro-apoptotic BH3-only proteins and Bcl-2/Bcl-XL. Since most of the patients with MM overexpress Bcl-2 protein, we considered Gos might be a promising therapeutic agent for MM. We herein show that Gos efficiently induced apoptosis and inhibited proliferation of the OPM2 MM cell line, in a dose- and time-dependent manner. Gos induced activation of caspase-3 and cytochrome c release from mitochondria, showing mitochondrial dysfunction pathway is operational during apoptosis. Further investigation revealed that phosphorylation of Bcl-2 at serine-70 was attenuated by Gos treatment, while protein levels were not affected. In addition, Mcl-1 was downregulated by Gos. Interestingly, phosphorylation of JAK2, STAT3, ERK1/2 and p38MAPK was inhibited by Gos-treatment, indicating that Gos globally suppressed interleukin-6 (IL-6) signals. Moreover, JAK2 inhibition mimicked the effect of Gos in OPM2 cells including Bcl-2 dephosphorylation and Mcl-1 downregulation. These results demonstrated that Gos induces apoptosis in MM cells not only through displacing BH3-only proteins from Bcl-2, but also through inhibiting IL-6 signaling, which leads to Bcl-2 dephosphorylation and Mcl-1 downregulation.

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Related in: MedlinePlus

Gossypol activates mitochondrial dysfunction pathway of apoptosis. (A) Flow cytometric analysis of mitochondrial transmembrane potential (ΔΨm). OPM2 cells were treated with 5 μM of Gos for 0–12 h, and mitochondrial membrane potential was measured by DioC6 fluorescence. (B) Release of cytochrome c proteins from mitochondria. Cytoplasmic or mitochondrial proteins were extracted from OPM2 cells treated with 5 μM of Gos for 0–24 h using Mitochondria/Cytosol Fractionation kit. β-actin was blotted as a loading control.
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f4-ijo-45-06-2278: Gossypol activates mitochondrial dysfunction pathway of apoptosis. (A) Flow cytometric analysis of mitochondrial transmembrane potential (ΔΨm). OPM2 cells were treated with 5 μM of Gos for 0–12 h, and mitochondrial membrane potential was measured by DioC6 fluorescence. (B) Release of cytochrome c proteins from mitochondria. Cytoplasmic or mitochondrial proteins were extracted from OPM2 cells treated with 5 μM of Gos for 0–24 h using Mitochondria/Cytosol Fractionation kit. β-actin was blotted as a loading control.

Mentions: To examine the activation of mitochondrial apoptosis pathway by Gos, we next examined the mitochondrial changes evoked by Gos treatment. Mitochondrial changes during apoptosis include permeability transition pore opening and the collapse of the mitochondrial transmembrane potential (ΔΨm), which results in the release of cytochrome c into the cytosol and subsequent activation of caspases. In order to examine these processes, we measured changes of mitochondrial ΔΨm in OPM2 cells treated with Gos by flow cytometry using DioC6. By treatment with 5 μM of Gos, ΔΨm of mitochondria decreased in a time-dependent manner in OPM2 cells (Fig. 4A). Furthermore, the decrease of ΔΨm was accompanied by the release of cytochrome c from mitochondria to cytosol (Fig. 4B). These results suggested that Gos-induced apoptosis is, at least in part, mediated through a mitochondria-dependent pathway.


Gossypol induces apoptosis in multiple myeloma cells by inhibition of interleukin-6 signaling and Bcl-2/Mcl-1 pathway.

Sadahira K, Sagawa M, Nakazato T, Uchida H, Ikeda Y, Okamoto S, Nakajima H, Kizaki M - Int. J. Oncol. (2014)

Gossypol activates mitochondrial dysfunction pathway of apoptosis. (A) Flow cytometric analysis of mitochondrial transmembrane potential (ΔΨm). OPM2 cells were treated with 5 μM of Gos for 0–12 h, and mitochondrial membrane potential was measured by DioC6 fluorescence. (B) Release of cytochrome c proteins from mitochondria. Cytoplasmic or mitochondrial proteins were extracted from OPM2 cells treated with 5 μM of Gos for 0–24 h using Mitochondria/Cytosol Fractionation kit. β-actin was blotted as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215583&req=5

f4-ijo-45-06-2278: Gossypol activates mitochondrial dysfunction pathway of apoptosis. (A) Flow cytometric analysis of mitochondrial transmembrane potential (ΔΨm). OPM2 cells were treated with 5 μM of Gos for 0–12 h, and mitochondrial membrane potential was measured by DioC6 fluorescence. (B) Release of cytochrome c proteins from mitochondria. Cytoplasmic or mitochondrial proteins were extracted from OPM2 cells treated with 5 μM of Gos for 0–24 h using Mitochondria/Cytosol Fractionation kit. β-actin was blotted as a loading control.
Mentions: To examine the activation of mitochondrial apoptosis pathway by Gos, we next examined the mitochondrial changes evoked by Gos treatment. Mitochondrial changes during apoptosis include permeability transition pore opening and the collapse of the mitochondrial transmembrane potential (ΔΨm), which results in the release of cytochrome c into the cytosol and subsequent activation of caspases. In order to examine these processes, we measured changes of mitochondrial ΔΨm in OPM2 cells treated with Gos by flow cytometry using DioC6. By treatment with 5 μM of Gos, ΔΨm of mitochondria decreased in a time-dependent manner in OPM2 cells (Fig. 4A). Furthermore, the decrease of ΔΨm was accompanied by the release of cytochrome c from mitochondria to cytosol (Fig. 4B). These results suggested that Gos-induced apoptosis is, at least in part, mediated through a mitochondria-dependent pathway.

Bottom Line: In addition, Mcl-1 was downregulated by Gos.Moreover, JAK2 inhibition mimicked the effect of Gos in OPM2 cells including Bcl-2 dephosphorylation and Mcl-1 downregulation.These results demonstrated that Gos induces apoptosis in MM cells not only through displacing BH3-only proteins from Bcl-2, but also through inhibiting IL-6 signaling, which leads to Bcl-2 dephosphorylation and Mcl-1 downregulation.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Department of Internal Medicine, Keio University School of Medicine, Tokyo 160-8582, Japan.

ABSTRACT
Multiple myeloma (MM) is a clonal plasma cell disorder affecting the immune system with various systemic symptoms. MM remains incurable even with high dose chemotherapy using conventional drugs, thus necessitating development of novel therapeutic strategies. Gossypol (Gos) is a natural polyphenolic compound extracted from cotton plants, and has been shown to possess anti-neoplastic activity against various tumors. Recent studies have shown that Gos is an inhibitor for Bcl-2 or Bcl-XL acting as BH3 mimetics that interfere interaction between pro-apoptotic BH3-only proteins and Bcl-2/Bcl-XL. Since most of the patients with MM overexpress Bcl-2 protein, we considered Gos might be a promising therapeutic agent for MM. We herein show that Gos efficiently induced apoptosis and inhibited proliferation of the OPM2 MM cell line, in a dose- and time-dependent manner. Gos induced activation of caspase-3 and cytochrome c release from mitochondria, showing mitochondrial dysfunction pathway is operational during apoptosis. Further investigation revealed that phosphorylation of Bcl-2 at serine-70 was attenuated by Gos treatment, while protein levels were not affected. In addition, Mcl-1 was downregulated by Gos. Interestingly, phosphorylation of JAK2, STAT3, ERK1/2 and p38MAPK was inhibited by Gos-treatment, indicating that Gos globally suppressed interleukin-6 (IL-6) signals. Moreover, JAK2 inhibition mimicked the effect of Gos in OPM2 cells including Bcl-2 dephosphorylation and Mcl-1 downregulation. These results demonstrated that Gos induces apoptosis in MM cells not only through displacing BH3-only proteins from Bcl-2, but also through inhibiting IL-6 signaling, which leads to Bcl-2 dephosphorylation and Mcl-1 downregulation.

Show MeSH
Related in: MedlinePlus