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Curcumin Prevents Palmitoylation of Integrin β4 in Breast Cancer Cells.

Coleman DT, Soung YH, Surh YJ, Cardelli JA, Chung J - PLoS ONE (2015)

Bottom Line: We found that the levels of ITG β4 palmitoylation correlated with the invasive potential of breast cancer cells, and that curcumin effectively reduced the levels of ITG β4 palmitoylation in invasive breast cancer cells.Through studies of ITG β4 palmitoylation kinetics, we concluded curcumin suppressed palmitoylation independent of growth factor-induced phosphorylation of key ITG β4 Ser and Tyr residues.Our studies reveal a novel paradigm for curcumin to account for much of its biological activity, and specifically, how it is able to suppress the signaling function of ITG β4 in breast cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and FeistWeiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana, United States of America.

ABSTRACT
Curcumin has been shown to mitigate cancer phenotypes such as invasive migration, proliferation, and survival by disrupting numerous signaling pathways. Our previous studies showed that curcumin inhibits integrin β4 (ITG β4)-dependent migration by blocking interaction of this integrin with growth factor receptors in lipid rafts. In the current study, we investigated the possibility that curcumin inhibits ITG β4 palmitoylation, a post-translational modification required for its lipid raft localization and signaling activity. We found that the levels of ITG β4 palmitoylation correlated with the invasive potential of breast cancer cells, and that curcumin effectively reduced the levels of ITG β4 palmitoylation in invasive breast cancer cells. Through studies of ITG β4 palmitoylation kinetics, we concluded curcumin suppressed palmitoylation independent of growth factor-induced phosphorylation of key ITG β4 Ser and Tyr residues. Rather, curcumin blocked autoacylation of the palmitoyl acyltransferase DHHC3 that is responsible for ITG β4 palmitoylation. Moreover, these data reveal that curcumin is able to prevent the palmitoylation of a subset of proteins, but not indiscriminately bind to and block all cysteines from modifications. Our studies reveal a novel paradigm for curcumin to account for much of its biological activity, and specifically, how it is able to suppress the signaling function of ITG β4 in breast cancer cells.

No MeSH data available.


Related in: MedlinePlus

Curcumin inhibits palmitoylation of ITG β4.(A) MDA-MB-231 cells were pretreated with 15 μM curcumin or 100 μM 2-BP (2-bromopalmitate) for 1hr prior to 17-ODYA labeling for 4 hr in the presence of either compound. (B) Cells were either treated with 15 μM curcumin1 hr before and during 17-ODYA labeling for 3 hours, or labeled with 17-ODYA for 3 hours followed by wash and 4 hours chase treatment with 15 μM curcumin. Labeled proteins were reacted to biotin-azide via click chemistry and biotinylated proteins were isolated using streptavidin-sepharose. Palmitoylated ITG β4 was detected by western blot analysis using anti-β4 antibody. Representative blots of 3 independent experiments are displayed with relative input protein included. Densitometry is shown in arbitrary units.
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pone.0125399.g001: Curcumin inhibits palmitoylation of ITG β4.(A) MDA-MB-231 cells were pretreated with 15 μM curcumin or 100 μM 2-BP (2-bromopalmitate) for 1hr prior to 17-ODYA labeling for 4 hr in the presence of either compound. (B) Cells were either treated with 15 μM curcumin1 hr before and during 17-ODYA labeling for 3 hours, or labeled with 17-ODYA for 3 hours followed by wash and 4 hours chase treatment with 15 μM curcumin. Labeled proteins were reacted to biotin-azide via click chemistry and biotinylated proteins were isolated using streptavidin-sepharose. Palmitoylated ITG β4 was detected by western blot analysis using anti-β4 antibody. Representative blots of 3 independent experiments are displayed with relative input protein included. Densitometry is shown in arbitrary units.

Mentions: Our previous work showed that inhibition of ITG β4 signaling by curcumin is mediated through blocking its lipid raft localization, thereby reducing its interaction with signaling receptors such as EGFR [4, 5]. However, a detailed molecular mechanism by which curcumin inhibits the mobilization of ITG β4 into the lipid rafts remains to be determined. Based on the previous report that the lipid raft localization of ITG β4 requires palmitoylation of transmembrane proximal cysteine residues [8], we tested whether curcumin affected palmitoylation of ITG β4. MDA-MB-231 cells were metabolically labeling with or without the alkyne-linked palmitate analogue, 17-ODYA [16,17], in the presence or absence of curcumin for 5 hours. Lysates were processed through a click chemistry-based alkyne to biotin conjugation and streptavidin-based pull-down assay to measure protein palmitoylation. As shown in Fig 1A, the baseline palmitoylation level of ITG β4 was detected in MDA-MB-231 breast cancer cells, and curcumin reduced ITG β4 palmitoylation similar to the level observed in cells treated with the palmitoylation inhibitor 2-bromopalmitate (2BP). For each experiment, a no ODYA control is included to indicate the non-specific background intensity following the click chemistry reaction and streptavidin-based pull-down. In order to determine if curcumin was able to reverse palmitoylation or simply block incorporation of labeled palmitate, cells were pulsed and chased with or without 17-ODYA in the presence or absence of curcumin. The results indicated that curcumin was consistently able to block palmitoylation of ITG β4, but was not able to reverse the attachment of labeled palmitate (Fig 1B).


Curcumin Prevents Palmitoylation of Integrin β4 in Breast Cancer Cells.

Coleman DT, Soung YH, Surh YJ, Cardelli JA, Chung J - PLoS ONE (2015)

Curcumin inhibits palmitoylation of ITG β4.(A) MDA-MB-231 cells were pretreated with 15 μM curcumin or 100 μM 2-BP (2-bromopalmitate) for 1hr prior to 17-ODYA labeling for 4 hr in the presence of either compound. (B) Cells were either treated with 15 μM curcumin1 hr before and during 17-ODYA labeling for 3 hours, or labeled with 17-ODYA for 3 hours followed by wash and 4 hours chase treatment with 15 μM curcumin. Labeled proteins were reacted to biotin-azide via click chemistry and biotinylated proteins were isolated using streptavidin-sepharose. Palmitoylated ITG β4 was detected by western blot analysis using anti-β4 antibody. Representative blots of 3 independent experiments are displayed with relative input protein included. Densitometry is shown in arbitrary units.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418632&req=5

pone.0125399.g001: Curcumin inhibits palmitoylation of ITG β4.(A) MDA-MB-231 cells were pretreated with 15 μM curcumin or 100 μM 2-BP (2-bromopalmitate) for 1hr prior to 17-ODYA labeling for 4 hr in the presence of either compound. (B) Cells were either treated with 15 μM curcumin1 hr before and during 17-ODYA labeling for 3 hours, or labeled with 17-ODYA for 3 hours followed by wash and 4 hours chase treatment with 15 μM curcumin. Labeled proteins were reacted to biotin-azide via click chemistry and biotinylated proteins were isolated using streptavidin-sepharose. Palmitoylated ITG β4 was detected by western blot analysis using anti-β4 antibody. Representative blots of 3 independent experiments are displayed with relative input protein included. Densitometry is shown in arbitrary units.
Mentions: Our previous work showed that inhibition of ITG β4 signaling by curcumin is mediated through blocking its lipid raft localization, thereby reducing its interaction with signaling receptors such as EGFR [4, 5]. However, a detailed molecular mechanism by which curcumin inhibits the mobilization of ITG β4 into the lipid rafts remains to be determined. Based on the previous report that the lipid raft localization of ITG β4 requires palmitoylation of transmembrane proximal cysteine residues [8], we tested whether curcumin affected palmitoylation of ITG β4. MDA-MB-231 cells were metabolically labeling with or without the alkyne-linked palmitate analogue, 17-ODYA [16,17], in the presence or absence of curcumin for 5 hours. Lysates were processed through a click chemistry-based alkyne to biotin conjugation and streptavidin-based pull-down assay to measure protein palmitoylation. As shown in Fig 1A, the baseline palmitoylation level of ITG β4 was detected in MDA-MB-231 breast cancer cells, and curcumin reduced ITG β4 palmitoylation similar to the level observed in cells treated with the palmitoylation inhibitor 2-bromopalmitate (2BP). For each experiment, a no ODYA control is included to indicate the non-specific background intensity following the click chemistry reaction and streptavidin-based pull-down. In order to determine if curcumin was able to reverse palmitoylation or simply block incorporation of labeled palmitate, cells were pulsed and chased with or without 17-ODYA in the presence or absence of curcumin. The results indicated that curcumin was consistently able to block palmitoylation of ITG β4, but was not able to reverse the attachment of labeled palmitate (Fig 1B).

Bottom Line: We found that the levels of ITG β4 palmitoylation correlated with the invasive potential of breast cancer cells, and that curcumin effectively reduced the levels of ITG β4 palmitoylation in invasive breast cancer cells.Through studies of ITG β4 palmitoylation kinetics, we concluded curcumin suppressed palmitoylation independent of growth factor-induced phosphorylation of key ITG β4 Ser and Tyr residues.Our studies reveal a novel paradigm for curcumin to account for much of its biological activity, and specifically, how it is able to suppress the signaling function of ITG β4 in breast cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and FeistWeiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana, United States of America.

ABSTRACT
Curcumin has been shown to mitigate cancer phenotypes such as invasive migration, proliferation, and survival by disrupting numerous signaling pathways. Our previous studies showed that curcumin inhibits integrin β4 (ITG β4)-dependent migration by blocking interaction of this integrin with growth factor receptors in lipid rafts. In the current study, we investigated the possibility that curcumin inhibits ITG β4 palmitoylation, a post-translational modification required for its lipid raft localization and signaling activity. We found that the levels of ITG β4 palmitoylation correlated with the invasive potential of breast cancer cells, and that curcumin effectively reduced the levels of ITG β4 palmitoylation in invasive breast cancer cells. Through studies of ITG β4 palmitoylation kinetics, we concluded curcumin suppressed palmitoylation independent of growth factor-induced phosphorylation of key ITG β4 Ser and Tyr residues. Rather, curcumin blocked autoacylation of the palmitoyl acyltransferase DHHC3 that is responsible for ITG β4 palmitoylation. Moreover, these data reveal that curcumin is able to prevent the palmitoylation of a subset of proteins, but not indiscriminately bind to and block all cysteines from modifications. Our studies reveal a novel paradigm for curcumin to account for much of its biological activity, and specifically, how it is able to suppress the signaling function of ITG β4 in breast cancer cells.

No MeSH data available.


Related in: MedlinePlus