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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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The 1D SDS-PAGE gel image of proteins from untreated and 24 h, 40 μmol/L gossypol-treated multiple myeloma cells. Lane 1: proteins from untreated cells cultured in the SILAC medium were mixed with proteins from gossypol-treated cells cultured in regular medium; and Lane 2: molecular weight markers.
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fig3: The 1D SDS-PAGE gel image of proteins from untreated and 24 h, 40 μmol/L gossypol-treated multiple myeloma cells. Lane 1: proteins from untreated cells cultured in the SILAC medium were mixed with proteins from gossypol-treated cells cultured in regular medium; and Lane 2: molecular weight markers.

Mentions: Next, proteomic analysis was carried out on the necrotic multiple myeloma cells. An equal amount of proteins (30 μg) from untreated and gossypol-treated U266 cells was mixed and separated by 1D SDS-PAGE (Figure 3). Differentially expressed proteins were identified and quantified using SILAC quantitation. The experiments were repeated twice and we identified about 4330 proteins. The false-positive rate was estimated to be less than 1%. Based on SILAC ratios (>1.6 or <0.7) and the protein scores (>10), 585 proteins were found to be differentially expressed between untreated and gossypol-treated cells, in which 383 proteins are downregulated and 202 are upregulated (see Supplementary Tables 1 and 2 in Supplementary Material available online at http://dx.doi.org/10.1155/2014/839232). In order to understand the biological relevance of the identified proteins, the gene ontology (GO) was used to cluster the differentially expressed proteins according to their molecular functions and biological processes. The annotations of gene lists are summarized via a pie plot using the PANTHER bioinformatics platform (http://www.pantherdb.org/) as shown in Figure 4. 166 proteins were classified into several significant groups of biological processes according to their molecular functions including energy and carbohydrate metabolism, RNA processing and protein synthesis, DNA repair, chaperone, and cell cycle regulation.


Proteomic analysis of gossypol induces necrosis in multiple myeloma cells.

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

The 1D SDS-PAGE gel image of proteins from untreated and 24 h, 40 μmol/L gossypol-treated multiple myeloma cells. Lane 1: proteins from untreated cells cultured in the SILAC medium were mixed with proteins from gossypol-treated cells cultured in regular medium; and Lane 2: molecular weight markers.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: The 1D SDS-PAGE gel image of proteins from untreated and 24 h, 40 μmol/L gossypol-treated multiple myeloma cells. Lane 1: proteins from untreated cells cultured in the SILAC medium were mixed with proteins from gossypol-treated cells cultured in regular medium; and Lane 2: molecular weight markers.
Mentions: Next, proteomic analysis was carried out on the necrotic multiple myeloma cells. An equal amount of proteins (30 μg) from untreated and gossypol-treated U266 cells was mixed and separated by 1D SDS-PAGE (Figure 3). Differentially expressed proteins were identified and quantified using SILAC quantitation. The experiments were repeated twice and we identified about 4330 proteins. The false-positive rate was estimated to be less than 1%. Based on SILAC ratios (>1.6 or <0.7) and the protein scores (>10), 585 proteins were found to be differentially expressed between untreated and gossypol-treated cells, in which 383 proteins are downregulated and 202 are upregulated (see Supplementary Tables 1 and 2 in Supplementary Material available online at http://dx.doi.org/10.1155/2014/839232). In order to understand the biological relevance of the identified proteins, the gene ontology (GO) was used to cluster the differentially expressed proteins according to their molecular functions and biological processes. The annotations of gene lists are summarized via a pie plot using the PANTHER bioinformatics platform (http://www.pantherdb.org/) as shown in Figure 4. 166 proteins were classified into several significant groups of biological processes according to their molecular functions including energy and carbohydrate metabolism, RNA processing and protein synthesis, DNA repair, chaperone, and cell cycle regulation.

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