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Proteomic analysis of gossypol induces necrosis in multiple myeloma cells.

Xu R, Tian E, Tang H, Liu C, Wang Q - Biomed Res Int (2014)

Bottom Line: Proteomic analysis identified 4330 proteins, in which 202 proteins are upregulated and 383 proteins are downregulated in gossypol-treated cells as compared to the untreated cells.Importantly, proteomic and western blot analysis showed that apoptosis regulators BAK and Bax were upregulated in gossypol-treated cells, indicating that Bcl-2 associated death pathway was activated.Similarly, gossypol also induced upregulations of DNA mismatch repair proteins and DNA replication licensing factor, suggesting that gossypol caused significant DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Clinical Laboratory of Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China ; Binzhou Medical University, Yantai 264003, China.

ABSTRACT
Gossypol is a phenolic aldehyde extracted from plants and is known to be an antitumor agent to induce cancer cell apoptosis. In the present study, multiple myeloma cells were treated with gossypol, which resulted in an increase of cellular reactive oxygen species (ROS) and cell necrosis. Quantitative proteomic analysis was carried out to identify differentially expressed proteins between untreated and gossypol-treated cells. Proteomic analysis identified 4330 proteins, in which 202 proteins are upregulated and 383 proteins are downregulated in gossypol-treated cells as compared to the untreated cells. Importantly, proteomic and western blot analysis showed that apoptosis regulators BAK and Bax were upregulated in gossypol-treated cells, indicating that Bcl-2 associated death pathway was activated. Similarly, gossypol also induced upregulations of DNA mismatch repair proteins and DNA replication licensing factor, suggesting that gossypol caused significant DNA damage. Furthermore, upregulations of HLA class I and class II histocompatibility antigens and beta-2-microglobulin were observed in gossypol-treated cells, indicating that gossypol has a novel function to activate cellular immune responses. Our data demonstrate that the execution of necrosis is a complex process involving ROS, DNA damage, and Bcl-2 family proteins. Gossypol-activated immune responses are a potential new approach for multiple myeloma chemotherapy.

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Western blot analysis of Bcl-2, Bax, caspase-3, HLA-A, HLA-DR, HSP60, NDUFA5, and P53 in untreated, 20 µmol/L and 40 µmol/L gossypol-treated cells. (a) Western blot image. (b) Analysis of western blots displayed in (a).
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fig5: Western blot analysis of Bcl-2, Bax, caspase-3, HLA-A, HLA-DR, HSP60, NDUFA5, and P53 in untreated, 20 µmol/L and 40 µmol/L gossypol-treated cells. (a) Western blot image. (b) Analysis of western blots displayed in (a).

Mentions: Using available quality antibodies, we carried out western blot analysis to quantify selected proteins that may play a role in gossypol-induced necrosis and to confirm selected proteins identified by proteomic analysis. Eight proteins were probed with antibodies and beta-actin was used as a control. Western blotting is displayed in Figure 5(a), showing that Bcl-2, Bax, caspase-3, HLA-A, HLA-DR, HSP60, and P53 are upregulated in gossypol-treated cells, while NADH dehydrogenase 1 alpha subcomplex 5 (NDUFA5) is downregulated. Quantitation of band intensities in western blot images was carried out using the Image Lab 4.0.1 Software (Figure 5(b)). The changes of the band intensities by western blotting are comparable to those determined by SILAC ratios for HLA-A and HLA-DR, indicating that quantitative proteomic analysis generates reliable results.


Proteomic analysis of gossypol induces necrosis in multiple myeloma cells.

Xu R, Tian E, Tang H, Liu C, Wang Q - Biomed Res Int (2014)

Western blot analysis of Bcl-2, Bax, caspase-3, HLA-A, HLA-DR, HSP60, NDUFA5, and P53 in untreated, 20 µmol/L and 40 µmol/L gossypol-treated cells. (a) Western blot image. (b) Analysis of western blots displayed in (a).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4150408&req=5

fig5: Western blot analysis of Bcl-2, Bax, caspase-3, HLA-A, HLA-DR, HSP60, NDUFA5, and P53 in untreated, 20 µmol/L and 40 µmol/L gossypol-treated cells. (a) Western blot image. (b) Analysis of western blots displayed in (a).
Mentions: Using available quality antibodies, we carried out western blot analysis to quantify selected proteins that may play a role in gossypol-induced necrosis and to confirm selected proteins identified by proteomic analysis. Eight proteins were probed with antibodies and beta-actin was used as a control. Western blotting is displayed in Figure 5(a), showing that Bcl-2, Bax, caspase-3, HLA-A, HLA-DR, HSP60, and P53 are upregulated in gossypol-treated cells, while NADH dehydrogenase 1 alpha subcomplex 5 (NDUFA5) is downregulated. Quantitation of band intensities in western blot images was carried out using the Image Lab 4.0.1 Software (Figure 5(b)). The changes of the band intensities by western blotting are comparable to those determined by SILAC ratios for HLA-A and HLA-DR, indicating that quantitative proteomic analysis generates reliable results.

Bottom Line: Proteomic analysis identified 4330 proteins, in which 202 proteins are upregulated and 383 proteins are downregulated in gossypol-treated cells as compared to the untreated cells.Importantly, proteomic and western blot analysis showed that apoptosis regulators BAK and Bax were upregulated in gossypol-treated cells, indicating that Bcl-2 associated death pathway was activated.Similarly, gossypol also induced upregulations of DNA mismatch repair proteins and DNA replication licensing factor, suggesting that gossypol caused significant DNA damage.

View Article: PubMed Central - PubMed

Affiliation: Clinical Laboratory of Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China ; Binzhou Medical University, Yantai 264003, China.

ABSTRACT
Gossypol is a phenolic aldehyde extracted from plants and is known to be an antitumor agent to induce cancer cell apoptosis. In the present study, multiple myeloma cells were treated with gossypol, which resulted in an increase of cellular reactive oxygen species (ROS) and cell necrosis. Quantitative proteomic analysis was carried out to identify differentially expressed proteins between untreated and gossypol-treated cells. Proteomic analysis identified 4330 proteins, in which 202 proteins are upregulated and 383 proteins are downregulated in gossypol-treated cells as compared to the untreated cells. Importantly, proteomic and western blot analysis showed that apoptosis regulators BAK and Bax were upregulated in gossypol-treated cells, indicating that Bcl-2 associated death pathway was activated. Similarly, gossypol also induced upregulations of DNA mismatch repair proteins and DNA replication licensing factor, suggesting that gossypol caused significant DNA damage. Furthermore, upregulations of HLA class I and class II histocompatibility antigens and beta-2-microglobulin were observed in gossypol-treated cells, indicating that gossypol has a novel function to activate cellular immune responses. Our data demonstrate that the execution of necrosis is a complex process involving ROS, DNA damage, and Bcl-2 family proteins. Gossypol-activated immune responses are a potential new approach for multiple myeloma chemotherapy.

Show MeSH
Related in: MedlinePlus