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Nutrient/serum starvation derived TRIP-Br3 down-regulation accelerates apoptosis by destabilizing XIAP.

Li C, Jung S, Lee S, Jeong D, Yang Y, Kim KI, Lim JS, Cheon CI, Kim C, Kang YS, Lee MS - Oncotarget (2015)

Bottom Line: However, the prolonged extreme stressful condition of nutrient starvation causes a dramatic decrease of TRIP-Br3, which in turn induces apoptosis by destabilizing XIAP.Up-regulated TRIP-Br1 in cancer cells compensates this effect and delays apoptosis.This can be explained by the competitive alternative binding of TRIP-Br3 and TRIP-Br1 to the BIR2 domain of XIAP.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Systems, Sookmyung Women's University, Seoul, 140-742, South Korea.

ABSTRACT
TRIP-Br3 and TRIP-Br1 have shown to have important biological functions. However, the function of TRIP-Br3 in tumorigenesis is not well characterized compared to oncogenic TRIP-Br1. Here, we investigated the function of TRIP-Br3 in tumorigenesis by comparing with that of TRIP-Br1. Under nutrient/serum starvation, TRIP-Br3 expression was down-regulated slightly in cancer cells and significantly in normal cells. Unexpectedly, TRIP-Br1 expression was greatly up-regulated in cancer cells but not in normal cells. Moreover, TRIP-Br3 activated autophagy while TRIP-Br1 inactivated it under serum starvation. In spite of different expression and roles of TRIP-Br3 and TRIP-Br1, both of them alleviate cell death by directly binding to and stabilizing XIAP, a potent apoptosis inhibitor, through blocking its ubiquitination. Taken together, we propose that TRIP-Br3 primarily activates the autophagy and suppresses apoptosis in nutrient sufficient condition. However, the prolonged extreme stressful condition of nutrient starvation causes a dramatic decrease of TRIP-Br3, which in turn induces apoptosis by destabilizing XIAP. Up-regulated TRIP-Br1 in cancer cells compensates this effect and delays apoptosis. This can be explained by the competitive alternative binding of TRIP-Br3 and TRIP-Br1 to the BIR2 domain of XIAP. In an extended study, our immunohistochemical analysis revealed a markedly lower level of TRIP-Br3 protein in human carcinoma tissues compared to normal epithelial tissues, implying the role of TRIP-Br3 as a tumor suppressor rather than onco-protein.

No MeSH data available.


Related in: MedlinePlus

Change of TRIP-Br3 expression level in different sub-cellular localizations under serum starvation(A) MCF7 and MCF10A cells were grown in media with or without serum for 48 h. Resulting cells were prepared for immunofluorescence and TRIP-Br3 localization was detected by using confocal microscopy. (B) Analysis of TRIP-Br3 location by nuclear fractionation in response to serum starvation for indicated times. Lamin B and γ-tubulin were used as nuclear marker or a loading control, respectively. Loaded amount of proteins are numbered. (C) Cells were grown in complete (CM) or serum starved (SS) medium in the absence or presence of 10 ng/ml of LMB for 36 h or 0.1 μM of MG132 for 20 h. Resulting cells were subjected to Western blot analysis. (D) TRIP-Br3 gene expression level was checked at the transcriptional level by employing RT-PCR. It was performed after indicated cells were subjected to serum containing complete medium (CM) or serum starved condition (SS) for 12 h or 24 h as mentioned in Methods and Materials. (E) Effect of PI3K/AKT and p38/MAPK on TRIP-Br3 expression under the condition of serum deficiencyEach cell line was grown in DMEM or DMEM/F12 with or without 50 μM of LY294002 or 20 μM of SB203580 for 24–48 h in the complete (CM) or serum starved (SS) media. (F) Cells were grown in DMEM or DMEM/F12 in the presence or absence of 20 μg/ml of insulin and/or 50 μM of EGF for 48 h in the complete (CM) or serum starved (SS) media.
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Figure 2: Change of TRIP-Br3 expression level in different sub-cellular localizations under serum starvation(A) MCF7 and MCF10A cells were grown in media with or without serum for 48 h. Resulting cells were prepared for immunofluorescence and TRIP-Br3 localization was detected by using confocal microscopy. (B) Analysis of TRIP-Br3 location by nuclear fractionation in response to serum starvation for indicated times. Lamin B and γ-tubulin were used as nuclear marker or a loading control, respectively. Loaded amount of proteins are numbered. (C) Cells were grown in complete (CM) or serum starved (SS) medium in the absence or presence of 10 ng/ml of LMB for 36 h or 0.1 μM of MG132 for 20 h. Resulting cells were subjected to Western blot analysis. (D) TRIP-Br3 gene expression level was checked at the transcriptional level by employing RT-PCR. It was performed after indicated cells were subjected to serum containing complete medium (CM) or serum starved condition (SS) for 12 h or 24 h as mentioned in Methods and Materials. (E) Effect of PI3K/AKT and p38/MAPK on TRIP-Br3 expression under the condition of serum deficiencyEach cell line was grown in DMEM or DMEM/F12 with or without 50 μM of LY294002 or 20 μM of SB203580 for 24–48 h in the complete (CM) or serum starved (SS) media. (F) Cells were grown in DMEM or DMEM/F12 in the presence or absence of 20 μg/ml of insulin and/or 50 μM of EGF for 48 h in the complete (CM) or serum starved (SS) media.

Mentions: To elucidate how TRIP-Br3 protein level is decreased under serum starvation, their cellular localization was at first checked. In the complete medium with sufficient serum, TRIP-Br3 was found in both nucleus and cytoplasm of MCF7 cells, but rarely found in nucleus of MCF10A cells (Figure 2A). In the serum starved medium, TRIP-Br3 signals became much weaker compared to those in normal condition (Figure 2A). This was confirmed by employing nuclear fractionation. TRIP-Br3 proteins were significantly decreased in the cytosol and/or nuclei of MCF7 and MCF10A cells in response to serum starvation (Figure 2B). It was proposed that TRIP-Br2 is localized in nucleus but mainly in cytoplasm, in which TRIP-Br2 is transported from nucleus into the cytoplasm through interaction with CRM1 and degraded by proteasome 26S [19, 22]. We therefore suspected that TRIP-Br3 might be transported and degraded in a similar way with that of TRIP-Br2. As expected, Leptomycin B (LMB, an inhibitor of the CRM1-dependent nuclear export pathway) treatment caused TRIP-Br3 to be retained in the nuclei of MCF7 and even MCF10A cells, in which TRIP-Br3 was not degraded (Figure 2A and 2C). In addition, MG132 (proteasome inhibitor) treatment slightly alleviated the TRIP-Br3 down-regulation under serum starvation. In a further study, RT-PCR analysis showed that TRIP-Br3 gene expression was not changed at the transcriptional level (Figure 2D). Taken together, our data suggest that in cancer cells, TRIP-Br3 is found in both cytoplasm and nucleus, in which nuclear TRIP-Br3 is exported to cytosol in a CRM1-dependent way and degraded at least partly by proteasome in response to serum deprivation. In normal cells, TRIP-Br3 presents mainly in the cytosol where cytosolic TRIP-Br3 more rapidly and greatly degraded by proteasome in response to serum starvation.


Nutrient/serum starvation derived TRIP-Br3 down-regulation accelerates apoptosis by destabilizing XIAP.

Li C, Jung S, Lee S, Jeong D, Yang Y, Kim KI, Lim JS, Cheon CI, Kim C, Kang YS, Lee MS - Oncotarget (2015)

Change of TRIP-Br3 expression level in different sub-cellular localizations under serum starvation(A) MCF7 and MCF10A cells were grown in media with or without serum for 48 h. Resulting cells were prepared for immunofluorescence and TRIP-Br3 localization was detected by using confocal microscopy. (B) Analysis of TRIP-Br3 location by nuclear fractionation in response to serum starvation for indicated times. Lamin B and γ-tubulin were used as nuclear marker or a loading control, respectively. Loaded amount of proteins are numbered. (C) Cells were grown in complete (CM) or serum starved (SS) medium in the absence or presence of 10 ng/ml of LMB for 36 h or 0.1 μM of MG132 for 20 h. Resulting cells were subjected to Western blot analysis. (D) TRIP-Br3 gene expression level was checked at the transcriptional level by employing RT-PCR. It was performed after indicated cells were subjected to serum containing complete medium (CM) or serum starved condition (SS) for 12 h or 24 h as mentioned in Methods and Materials. (E) Effect of PI3K/AKT and p38/MAPK on TRIP-Br3 expression under the condition of serum deficiencyEach cell line was grown in DMEM or DMEM/F12 with or without 50 μM of LY294002 or 20 μM of SB203580 for 24–48 h in the complete (CM) or serum starved (SS) media. (F) Cells were grown in DMEM or DMEM/F12 in the presence or absence of 20 μg/ml of insulin and/or 50 μM of EGF for 48 h in the complete (CM) or serum starved (SS) media.
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Related In: Results  -  Collection

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Figure 2: Change of TRIP-Br3 expression level in different sub-cellular localizations under serum starvation(A) MCF7 and MCF10A cells were grown in media with or without serum for 48 h. Resulting cells were prepared for immunofluorescence and TRIP-Br3 localization was detected by using confocal microscopy. (B) Analysis of TRIP-Br3 location by nuclear fractionation in response to serum starvation for indicated times. Lamin B and γ-tubulin were used as nuclear marker or a loading control, respectively. Loaded amount of proteins are numbered. (C) Cells were grown in complete (CM) or serum starved (SS) medium in the absence or presence of 10 ng/ml of LMB for 36 h or 0.1 μM of MG132 for 20 h. Resulting cells were subjected to Western blot analysis. (D) TRIP-Br3 gene expression level was checked at the transcriptional level by employing RT-PCR. It was performed after indicated cells were subjected to serum containing complete medium (CM) or serum starved condition (SS) for 12 h or 24 h as mentioned in Methods and Materials. (E) Effect of PI3K/AKT and p38/MAPK on TRIP-Br3 expression under the condition of serum deficiencyEach cell line was grown in DMEM or DMEM/F12 with or without 50 μM of LY294002 or 20 μM of SB203580 for 24–48 h in the complete (CM) or serum starved (SS) media. (F) Cells were grown in DMEM or DMEM/F12 in the presence or absence of 20 μg/ml of insulin and/or 50 μM of EGF for 48 h in the complete (CM) or serum starved (SS) media.
Mentions: To elucidate how TRIP-Br3 protein level is decreased under serum starvation, their cellular localization was at first checked. In the complete medium with sufficient serum, TRIP-Br3 was found in both nucleus and cytoplasm of MCF7 cells, but rarely found in nucleus of MCF10A cells (Figure 2A). In the serum starved medium, TRIP-Br3 signals became much weaker compared to those in normal condition (Figure 2A). This was confirmed by employing nuclear fractionation. TRIP-Br3 proteins were significantly decreased in the cytosol and/or nuclei of MCF7 and MCF10A cells in response to serum starvation (Figure 2B). It was proposed that TRIP-Br2 is localized in nucleus but mainly in cytoplasm, in which TRIP-Br2 is transported from nucleus into the cytoplasm through interaction with CRM1 and degraded by proteasome 26S [19, 22]. We therefore suspected that TRIP-Br3 might be transported and degraded in a similar way with that of TRIP-Br2. As expected, Leptomycin B (LMB, an inhibitor of the CRM1-dependent nuclear export pathway) treatment caused TRIP-Br3 to be retained in the nuclei of MCF7 and even MCF10A cells, in which TRIP-Br3 was not degraded (Figure 2A and 2C). In addition, MG132 (proteasome inhibitor) treatment slightly alleviated the TRIP-Br3 down-regulation under serum starvation. In a further study, RT-PCR analysis showed that TRIP-Br3 gene expression was not changed at the transcriptional level (Figure 2D). Taken together, our data suggest that in cancer cells, TRIP-Br3 is found in both cytoplasm and nucleus, in which nuclear TRIP-Br3 is exported to cytosol in a CRM1-dependent way and degraded at least partly by proteasome in response to serum deprivation. In normal cells, TRIP-Br3 presents mainly in the cytosol where cytosolic TRIP-Br3 more rapidly and greatly degraded by proteasome in response to serum starvation.

Bottom Line: However, the prolonged extreme stressful condition of nutrient starvation causes a dramatic decrease of TRIP-Br3, which in turn induces apoptosis by destabilizing XIAP.Up-regulated TRIP-Br1 in cancer cells compensates this effect and delays apoptosis.This can be explained by the competitive alternative binding of TRIP-Br3 and TRIP-Br1 to the BIR2 domain of XIAP.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Systems, Sookmyung Women's University, Seoul, 140-742, South Korea.

ABSTRACT
TRIP-Br3 and TRIP-Br1 have shown to have important biological functions. However, the function of TRIP-Br3 in tumorigenesis is not well characterized compared to oncogenic TRIP-Br1. Here, we investigated the function of TRIP-Br3 in tumorigenesis by comparing with that of TRIP-Br1. Under nutrient/serum starvation, TRIP-Br3 expression was down-regulated slightly in cancer cells and significantly in normal cells. Unexpectedly, TRIP-Br1 expression was greatly up-regulated in cancer cells but not in normal cells. Moreover, TRIP-Br3 activated autophagy while TRIP-Br1 inactivated it under serum starvation. In spite of different expression and roles of TRIP-Br3 and TRIP-Br1, both of them alleviate cell death by directly binding to and stabilizing XIAP, a potent apoptosis inhibitor, through blocking its ubiquitination. Taken together, we propose that TRIP-Br3 primarily activates the autophagy and suppresses apoptosis in nutrient sufficient condition. However, the prolonged extreme stressful condition of nutrient starvation causes a dramatic decrease of TRIP-Br3, which in turn induces apoptosis by destabilizing XIAP. Up-regulated TRIP-Br1 in cancer cells compensates this effect and delays apoptosis. This can be explained by the competitive alternative binding of TRIP-Br3 and TRIP-Br1 to the BIR2 domain of XIAP. In an extended study, our immunohistochemical analysis revealed a markedly lower level of TRIP-Br3 protein in human carcinoma tissues compared to normal epithelial tissues, implying the role of TRIP-Br3 as a tumor suppressor rather than onco-protein.

No MeSH data available.


Related in: MedlinePlus