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Cyanidin-3-glucoside inhibits ethanol-induced invasion of breast cancer cells overexpressing ErbB2.

Xu M, Bower KA, Wang S, Frank JA, Chen G, Ding M, Wang S, Shi X, Ke Z, Luo J - Mol. Cancer (2010)

Bottom Line: C3G decreased ethanol-mediated cell adhesion to the extracellular matrix (ECM) as well as the amount of focal adhesions and the formation of lamellipodial protrusion.It inhibited ethanol-stimulated phosphorylation of ErbB2, cSrc, FAK and p130(Cas), as well as interactions among these proteins.C3G may be beneficial in preventing/reducing ethanol-induced breast cancer metastasis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Internal Medicine, University of Kentucky College of Medicine, Lexington, KY 40536, USA.

ABSTRACT

Background: Ethanol is a tumor promoter. Both epidemiological and experimental studies suggest that ethanol may enhance the metastasis of breast cancer cells. We have previously demonstrated that ethanol increased the migration/invasion of breast cancer cells expressing high levels of ErbB2. Amplification of ErbB2 is found in 20-30% of breast cancer patients and is associated with poor prognosis. We sought to identify agents that can prevent or ameliorate ethanol-induced invasion of breast cancer cells. Cyanidin-3-glucoside (C3G), an anthocyanin present in many vegetables and fruits, is a potent natural antioxidant. Ethanol exposure causes the accumulation of intracellular reactive oxygen species (ROS). This study evaluated the effect of C3G on ethanol-induced breast cancer cell migration/invasion.

Results: C3G attenuated ethanol-induced migration/invasion of breast cancer cells expressing high levels of ErbB2 (BT474, MDA-MB231 and MCF7(ErbB2)) in a concentration dependent manner. C3G decreased ethanol-mediated cell adhesion to the extracellular matrix (ECM) as well as the amount of focal adhesions and the formation of lamellipodial protrusion. It inhibited ethanol-stimulated phosphorylation of ErbB2, cSrc, FAK and p130(Cas), as well as interactions among these proteins. C3G abolished ethanol-mediated p130(Cas)/JNK interaction.

Conclusions: C3G blocks ethanol-induced activation of the ErbB2/cSrc/FAK pathway which is necessary for cell migration/invasion. C3G may be beneficial in preventing/reducing ethanol-induced breast cancer metastasis.

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Effects of C3G on ethanol-mediated phosphorylation of ErbB2, cSrc, FAK and p130Cas. A: MCF7ErbB2 cells were treated with ethanol (0 or 400 mg/dl) with/without C3G (10, 20 or 40 μM) for 48 h. Cells were seeded on fibronectin-coated culture wells for 3 h and then harvested for analysis of the phosphorylation of ErbB2, FAK, p130Cas and cSrc with immunoblotting. The expression of actin served as a loading control. B: The relative expression of phosphorylated ErbB2, FAK, p130Cas and cSrc was determined by densitometry and normalized to the expression of actin. * denotes a statistically significant difference from untreated controls. # denotes a significant difference from ethanol-treated groups. ε denotes a significant difference from ethanol- and C3G (10 μM)-treated groups. δ denotes a significant difference from ethanol- and C3G (20 μM)-treated groups. C: The phosphorylation of ErbB2 and FAK in MDA-MB231 cells was analyzed as described above. The experiment was replicated three times.
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Figure 3: Effects of C3G on ethanol-mediated phosphorylation of ErbB2, cSrc, FAK and p130Cas. A: MCF7ErbB2 cells were treated with ethanol (0 or 400 mg/dl) with/without C3G (10, 20 or 40 μM) for 48 h. Cells were seeded on fibronectin-coated culture wells for 3 h and then harvested for analysis of the phosphorylation of ErbB2, FAK, p130Cas and cSrc with immunoblotting. The expression of actin served as a loading control. B: The relative expression of phosphorylated ErbB2, FAK, p130Cas and cSrc was determined by densitometry and normalized to the expression of actin. * denotes a statistically significant difference from untreated controls. # denotes a significant difference from ethanol-treated groups. ε denotes a significant difference from ethanol- and C3G (10 μM)-treated groups. δ denotes a significant difference from ethanol- and C3G (20 μM)-treated groups. C: The phosphorylation of ErbB2 and FAK in MDA-MB231 cells was analyzed as described above. The experiment was replicated three times.

Mentions: We have previously shown that ethanol increased the phosphorylation of ErbB2 at Tyr1248 [19]. In this study, we examined the effect of C3G on ethanol-mediated ErbB2 phosphorylation. MDA-MB231 and MCF7ErbB2 cells were pretreated with ethanol with/without C3G for 48 hours, then cells were seeded into fibronectin precoated dishes, allowing attachment for 3 hours. As shown in Figure 3, ethanol drastically increased the phosphorylation of ErbB2 [p-ErbB2(Tyr1248)] in these cells. The addition of C3G attenuated ethanol-stimulated p-ErbB2(Tyr1248) in a concentration-dependent manner. The cSrc/FAK pathway plays an important role in ErbB2-regulated migration/invasion of breast cancer cells [24]. FAK is a substrate of cSrc and FAK Tyr861 is a major site of phosphorylation by cSrc. As shown in Figure 3, ethanol increased the levels of p-FAK(Tyr861) and p-cSrc(Tyr216). C3G attenuated ethanol-induced p-FAK(Tyr861) and p-cSrc(Tyr216). The activation and phosphorylation of cSrc/FAK is critical for triggering its downstream signaling and for recruiting proteins to the focal adhesion sites. p130Cas, an adaptor protein, binds to the C-terminal site of FAK, forming a dock site for Crk; p130Cas/Crk interaction induces the activation of small GTPases and JNKs, promoting membrane protrusion and cell migration [25,26]. The phosphorylation of p130Cas is regulated by FAK and cSrc [27]. We demonstrated that ethanol induced the phosphorylation of p130Cas [p-p130Cas(Tyr410)], and C3G blocked ethanol-induced p-p130Cas(Tyr410) (Figure 3A). We further examined the effect of C3G on the interaction among ErbB2, cSrc, FAK and p130Cas. MCF7ErbB2 cells were treated with ethanol or with/without C3G for 48 hours and seeded on fibronectin for 1 or 3 hours. As shown in Figure 4, ethanol increased the association between ErbB2/FAK, FAK/cSrc, FAK/p130Cas and cSrc/p130Cas. C3G abolished the interaction among these proteins (Figure 4). These data indicated that C3G inhibited the ethanol-activated ErbB2/cSrc/FAK pathway.


Cyanidin-3-glucoside inhibits ethanol-induced invasion of breast cancer cells overexpressing ErbB2.

Xu M, Bower KA, Wang S, Frank JA, Chen G, Ding M, Wang S, Shi X, Ke Z, Luo J - Mol. Cancer (2010)

Effects of C3G on ethanol-mediated phosphorylation of ErbB2, cSrc, FAK and p130Cas. A: MCF7ErbB2 cells were treated with ethanol (0 or 400 mg/dl) with/without C3G (10, 20 or 40 μM) for 48 h. Cells were seeded on fibronectin-coated culture wells for 3 h and then harvested for analysis of the phosphorylation of ErbB2, FAK, p130Cas and cSrc with immunoblotting. The expression of actin served as a loading control. B: The relative expression of phosphorylated ErbB2, FAK, p130Cas and cSrc was determined by densitometry and normalized to the expression of actin. * denotes a statistically significant difference from untreated controls. # denotes a significant difference from ethanol-treated groups. ε denotes a significant difference from ethanol- and C3G (10 μM)-treated groups. δ denotes a significant difference from ethanol- and C3G (20 μM)-treated groups. C: The phosphorylation of ErbB2 and FAK in MDA-MB231 cells was analyzed as described above. The experiment was replicated three times.
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Figure 3: Effects of C3G on ethanol-mediated phosphorylation of ErbB2, cSrc, FAK and p130Cas. A: MCF7ErbB2 cells were treated with ethanol (0 or 400 mg/dl) with/without C3G (10, 20 or 40 μM) for 48 h. Cells were seeded on fibronectin-coated culture wells for 3 h and then harvested for analysis of the phosphorylation of ErbB2, FAK, p130Cas and cSrc with immunoblotting. The expression of actin served as a loading control. B: The relative expression of phosphorylated ErbB2, FAK, p130Cas and cSrc was determined by densitometry and normalized to the expression of actin. * denotes a statistically significant difference from untreated controls. # denotes a significant difference from ethanol-treated groups. ε denotes a significant difference from ethanol- and C3G (10 μM)-treated groups. δ denotes a significant difference from ethanol- and C3G (20 μM)-treated groups. C: The phosphorylation of ErbB2 and FAK in MDA-MB231 cells was analyzed as described above. The experiment was replicated three times.
Mentions: We have previously shown that ethanol increased the phosphorylation of ErbB2 at Tyr1248 [19]. In this study, we examined the effect of C3G on ethanol-mediated ErbB2 phosphorylation. MDA-MB231 and MCF7ErbB2 cells were pretreated with ethanol with/without C3G for 48 hours, then cells were seeded into fibronectin precoated dishes, allowing attachment for 3 hours. As shown in Figure 3, ethanol drastically increased the phosphorylation of ErbB2 [p-ErbB2(Tyr1248)] in these cells. The addition of C3G attenuated ethanol-stimulated p-ErbB2(Tyr1248) in a concentration-dependent manner. The cSrc/FAK pathway plays an important role in ErbB2-regulated migration/invasion of breast cancer cells [24]. FAK is a substrate of cSrc and FAK Tyr861 is a major site of phosphorylation by cSrc. As shown in Figure 3, ethanol increased the levels of p-FAK(Tyr861) and p-cSrc(Tyr216). C3G attenuated ethanol-induced p-FAK(Tyr861) and p-cSrc(Tyr216). The activation and phosphorylation of cSrc/FAK is critical for triggering its downstream signaling and for recruiting proteins to the focal adhesion sites. p130Cas, an adaptor protein, binds to the C-terminal site of FAK, forming a dock site for Crk; p130Cas/Crk interaction induces the activation of small GTPases and JNKs, promoting membrane protrusion and cell migration [25,26]. The phosphorylation of p130Cas is regulated by FAK and cSrc [27]. We demonstrated that ethanol induced the phosphorylation of p130Cas [p-p130Cas(Tyr410)], and C3G blocked ethanol-induced p-p130Cas(Tyr410) (Figure 3A). We further examined the effect of C3G on the interaction among ErbB2, cSrc, FAK and p130Cas. MCF7ErbB2 cells were treated with ethanol or with/without C3G for 48 hours and seeded on fibronectin for 1 or 3 hours. As shown in Figure 4, ethanol increased the association between ErbB2/FAK, FAK/cSrc, FAK/p130Cas and cSrc/p130Cas. C3G abolished the interaction among these proteins (Figure 4). These data indicated that C3G inhibited the ethanol-activated ErbB2/cSrc/FAK pathway.

Bottom Line: C3G decreased ethanol-mediated cell adhesion to the extracellular matrix (ECM) as well as the amount of focal adhesions and the formation of lamellipodial protrusion.It inhibited ethanol-stimulated phosphorylation of ErbB2, cSrc, FAK and p130(Cas), as well as interactions among these proteins.C3G may be beneficial in preventing/reducing ethanol-induced breast cancer metastasis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Internal Medicine, University of Kentucky College of Medicine, Lexington, KY 40536, USA.

ABSTRACT

Background: Ethanol is a tumor promoter. Both epidemiological and experimental studies suggest that ethanol may enhance the metastasis of breast cancer cells. We have previously demonstrated that ethanol increased the migration/invasion of breast cancer cells expressing high levels of ErbB2. Amplification of ErbB2 is found in 20-30% of breast cancer patients and is associated with poor prognosis. We sought to identify agents that can prevent or ameliorate ethanol-induced invasion of breast cancer cells. Cyanidin-3-glucoside (C3G), an anthocyanin present in many vegetables and fruits, is a potent natural antioxidant. Ethanol exposure causes the accumulation of intracellular reactive oxygen species (ROS). This study evaluated the effect of C3G on ethanol-induced breast cancer cell migration/invasion.

Results: C3G attenuated ethanol-induced migration/invasion of breast cancer cells expressing high levels of ErbB2 (BT474, MDA-MB231 and MCF7(ErbB2)) in a concentration dependent manner. C3G decreased ethanol-mediated cell adhesion to the extracellular matrix (ECM) as well as the amount of focal adhesions and the formation of lamellipodial protrusion. It inhibited ethanol-stimulated phosphorylation of ErbB2, cSrc, FAK and p130(Cas), as well as interactions among these proteins. C3G abolished ethanol-mediated p130(Cas)/JNK interaction.

Conclusions: C3G blocks ethanol-induced activation of the ErbB2/cSrc/FAK pathway which is necessary for cell migration/invasion. C3G may be beneficial in preventing/reducing ethanol-induced breast cancer metastasis.

Show MeSH
Related in: MedlinePlus