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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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Morphologic images of multiple myeloma cells. (a) Untreated cells; (b) 40 μmol/L gossypol-treated cells for 24 h; all images were captured by Olympus IX2-UCB 60x inverted microscopy; (c) gel electrophoresis of DNA from untreated and gossypol-treated myeloma cells. (d-e) Detection of ROS in untreated and gossypol-treated U266 cells using the Image-iT LIVE Reactive Oxygen Species (ROS) Kit. Cells were labeled with carboxy-H2DCFDA, which exhibited green fluorescence when reacted with ROS, and nuclei were stained with blue-fluorescent Hoechst 33342. (d) Fluorescence image of ROS in untreated U266 cells; and (e) fluorescence image of ROS in 40 μmol/L gossypol-treated cells for 10 h.
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fig2: Morphologic images of multiple myeloma cells. (a) Untreated cells; (b) 40 μmol/L gossypol-treated cells for 24 h; all images were captured by Olympus IX2-UCB 60x inverted microscopy; (c) gel electrophoresis of DNA from untreated and gossypol-treated myeloma cells. (d-e) Detection of ROS in untreated and gossypol-treated U266 cells using the Image-iT LIVE Reactive Oxygen Species (ROS) Kit. Cells were labeled with carboxy-H2DCFDA, which exhibited green fluorescence when reacted with ROS, and nuclei were stained with blue-fluorescent Hoechst 33342. (d) Fluorescence image of ROS in untreated U266 cells; and (e) fluorescence image of ROS in 40 μmol/L gossypol-treated cells for 10 h.

Mentions: FACS analysis showed that the percentage of necrotic cells was 22% when cells were treated with 20 μmol/L gossypol for 24 h, increasing to 82% when treated with 80 μmol/L gossypol for 24 h (Figure 1). Morphological features of the dying cells were consistent with the cell necrosis. Images of cell morphology in untreated and gossypol-treated cells are shown in Figures 2(a) and 2(b), respectively. The gossypol-treated multiple myeloma cells displayed characteristic features of necrosis, including cell swelling, translucent cytoplasm, cell membrane disruption, pyknotic nuclei, and excessive cellular debris. The DNA content of necrotic cells was analyzed by gel electrophoresis. The gel image of DNA for untreated and gossypol-treated cells (Figure 2(c)) shows that DNA from gossypol-treated cells exhibited a random and general cleavage pattern and produced a smear that further confirmed that gossypol-induced cell death occurs mainly via necrosis. The above data suggests that oxidative stress may cause necrosis in gossypol-treated cells. To confirm that ROS contributes to gossypol-induced cell necrosis, an Image-iT LIVE Reactive Oxygen Species (ROS) Kit was used to detect ROS in the untreated and gossypol-treated cells. Cells were labeled with carboxy-H2DCFDA, which fluoresces when oxidized by ROS, and nuclei were stained with blue-fluorescent Hoechst 33342. The gossypol-treated cells exhibited much stronger green fluorescence (Figure 2(e)) in comparison to untreated cells (Figure 2(d)), indicating that gossypol induced a significant increase in ROS production.


Proteomic analysis of gossypol induces necrosis in multiple myeloma cells.

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

Morphologic images of multiple myeloma cells. (a) Untreated cells; (b) 40 μmol/L gossypol-treated cells for 24 h; all images were captured by Olympus IX2-UCB 60x inverted microscopy; (c) gel electrophoresis of DNA from untreated and gossypol-treated myeloma cells. (d-e) Detection of ROS in untreated and gossypol-treated U266 cells using the Image-iT LIVE Reactive Oxygen Species (ROS) Kit. Cells were labeled with carboxy-H2DCFDA, which exhibited green fluorescence when reacted with ROS, and nuclei were stained with blue-fluorescent Hoechst 33342. (d) Fluorescence image of ROS in untreated U266 cells; and (e) fluorescence image of ROS in 40 μmol/L gossypol-treated cells for 10 h.
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fig2: Morphologic images of multiple myeloma cells. (a) Untreated cells; (b) 40 μmol/L gossypol-treated cells for 24 h; all images were captured by Olympus IX2-UCB 60x inverted microscopy; (c) gel electrophoresis of DNA from untreated and gossypol-treated myeloma cells. (d-e) Detection of ROS in untreated and gossypol-treated U266 cells using the Image-iT LIVE Reactive Oxygen Species (ROS) Kit. Cells were labeled with carboxy-H2DCFDA, which exhibited green fluorescence when reacted with ROS, and nuclei were stained with blue-fluorescent Hoechst 33342. (d) Fluorescence image of ROS in untreated U266 cells; and (e) fluorescence image of ROS in 40 μmol/L gossypol-treated cells for 10 h.
Mentions: FACS analysis showed that the percentage of necrotic cells was 22% when cells were treated with 20 μmol/L gossypol for 24 h, increasing to 82% when treated with 80 μmol/L gossypol for 24 h (Figure 1). Morphological features of the dying cells were consistent with the cell necrosis. Images of cell morphology in untreated and gossypol-treated cells are shown in Figures 2(a) and 2(b), respectively. The gossypol-treated multiple myeloma cells displayed characteristic features of necrosis, including cell swelling, translucent cytoplasm, cell membrane disruption, pyknotic nuclei, and excessive cellular debris. The DNA content of necrotic cells was analyzed by gel electrophoresis. The gel image of DNA for untreated and gossypol-treated cells (Figure 2(c)) shows that DNA from gossypol-treated cells exhibited a random and general cleavage pattern and produced a smear that further confirmed that gossypol-induced cell death occurs mainly via necrosis. The above data suggests that oxidative stress may cause necrosis in gossypol-treated cells. To confirm that ROS contributes to gossypol-induced cell necrosis, an Image-iT LIVE Reactive Oxygen Species (ROS) Kit was used to detect ROS in the untreated and gossypol-treated cells. Cells were labeled with carboxy-H2DCFDA, which fluoresces when oxidized by ROS, and nuclei were stained with blue-fluorescent Hoechst 33342. The gossypol-treated cells exhibited much stronger green fluorescence (Figure 2(e)) in comparison to untreated cells (Figure 2(d)), indicating that gossypol induced a significant increase in ROS production.

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