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Em08red, a dual functional antiproliferative emodin analogue, is a downregulator of ErbB2 expression and inducer of intracellular oxidative stress.

Liang FP, Lien JC, Wu YH, Chen CS, Juang SH - Drug Des Devel Ther (2015)

Bottom Line: Treatment with em08red significantly downregulated activation of ErbB2 as well as the ErbB2 protein expression level in the tested cell lines and induced G2 arrest.Antiapoptosis protein (Bcl-xl and Bcl-2) expression levels were also downregulated, and active caspase-3 and caspase-9 was detected in cells after treatment with em08red.Moreover, treatment with em08red stimulated production of cytotoxic reactive oxygen species in treated cells, and this could be partially reversed by pretreatment with N-acetylcysteine.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Pharmaceutical Chemistry, China Medical University Hospital, Taichung, Taiwan.

ABSTRACT
Expression of ErbB2 protein is inversely correlated with the prognosis in cancer patients. Consequently, strategies targeting ErbB2 remain an attractive option in treating several types of malignancies, including oral cancer. In addition, many studies have shown that emodin and emodin derivatives are able to inhibit growth of ErbB2-overexpressing tumor cells. In this study, a series of computer modeling-generated emodin analogues were synthesized and tested for their antiproliferative activity against oral cancer cell lines overexpressing ErbB2. Among these analogues, em08red (1,8-dihydroxy-9(10H)-anthracenone) demonstrated potent antiproliferative activity against all three tested ErbB2-overexpressing cell lines, ie, FaDu, HSC3, and OECM1. Treatment with em08red significantly downregulated activation of ErbB2 as well as the ErbB2 protein expression level in the tested cell lines and induced G2 arrest. Antiapoptosis protein (Bcl-xl and Bcl-2) expression levels were also downregulated, and active caspase-3 and caspase-9 was detected in cells after treatment with em08red. Moreover, treatment with em08red stimulated production of cytotoxic reactive oxygen species in treated cells, and this could be partially reversed by pretreatment with N-acetylcysteine. Overall, we demonstrated inhibition of ErbB2 function and induction of reactive oxygen species in tumor cells by em08red, which prevented proliferation of tumor cells and induced apoptotic cell death.

No MeSH data available.


Related in: MedlinePlus

Treatment with em08red induced G2 phase cell cycle arrest.Notes: (A) FaDu (left panel) and HSC3 (right panel) cells were treated with vehicle (0.1% dimethyl sulfoxide) or em08red (10 μM) for 24, 48, and 72 hours and then harvested for fixation. After staining with propidium iodide, cell cycle analysis was carried out using a Canto II cytometer. The data are representative of three independent experiments. (B) Treated FaDu cells were harvested at 12, 24, 36, and 48 hours, and total cell lysates were subjected to Western blot analysis. Antibodies specific for phosphor-Cdk1, total Cdk1, cyclin B, and phosphor-MPM2 were used to detect expression of each protein. Actin expression served as the loading control.
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f3-dddt-9-1499: Treatment with em08red induced G2 phase cell cycle arrest.Notes: (A) FaDu (left panel) and HSC3 (right panel) cells were treated with vehicle (0.1% dimethyl sulfoxide) or em08red (10 μM) for 24, 48, and 72 hours and then harvested for fixation. After staining with propidium iodide, cell cycle analysis was carried out using a Canto II cytometer. The data are representative of three independent experiments. (B) Treated FaDu cells were harvested at 12, 24, 36, and 48 hours, and total cell lysates were subjected to Western blot analysis. Antibodies specific for phosphor-Cdk1, total Cdk1, cyclin B, and phosphor-MPM2 were used to detect expression of each protein. Actin expression served as the loading control.

Mentions: A previous study showed that downregulation of ErbB2 protein expression by treatment with neu differential factor could induce arrest of tumor cells in G2/M phase in cells overexpressing ErbB2.21 To determine if em08red has a similar pharmacological effect on cell cycle progression, em08red-treated cells were subjected to flow cytometry analysis. The data indicated significant G2/M accumulation after 24 hours of treatment with em08red in both FaDu and HSC3 cell lines (Figure 3A). G2/M phase-associated proteins, Cdk1 and cyclin B,22 were downregulated after treatment with em08red and phosphor-MPM2 signals were also reduced after em08red, indicating that the treated cells were blocked at G2 phase (Figure 3B). Additionally, we observed a population of sub-G1 em08red-treated HSC3 and FaDu cells after 24 and 48 hours of treatment, respectively, indicative of G2 phase arrest and cell death (Figure 3A). To determine if the molecular mechanisms of em08red-induced cell death occurred via apoptosis or necrosis, the phosphatidylserine level (a marker of apoptosis) on the outer membrane of em08red-treated cells was examined by annexin V staining. As shown in Figure 4A, the percentage annexin V-positive population in em08red-treated FaDu and HSC3 cells increased in a time-dependent manner and correlated with our observations on sub-G1 cell cycle analysis.


Em08red, a dual functional antiproliferative emodin analogue, is a downregulator of ErbB2 expression and inducer of intracellular oxidative stress.

Liang FP, Lien JC, Wu YH, Chen CS, Juang SH - Drug Des Devel Ther (2015)

Treatment with em08red induced G2 phase cell cycle arrest.Notes: (A) FaDu (left panel) and HSC3 (right panel) cells were treated with vehicle (0.1% dimethyl sulfoxide) or em08red (10 μM) for 24, 48, and 72 hours and then harvested for fixation. After staining with propidium iodide, cell cycle analysis was carried out using a Canto II cytometer. The data are representative of three independent experiments. (B) Treated FaDu cells were harvested at 12, 24, 36, and 48 hours, and total cell lysates were subjected to Western blot analysis. Antibodies specific for phosphor-Cdk1, total Cdk1, cyclin B, and phosphor-MPM2 were used to detect expression of each protein. Actin expression served as the loading control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362900&req=5

f3-dddt-9-1499: Treatment with em08red induced G2 phase cell cycle arrest.Notes: (A) FaDu (left panel) and HSC3 (right panel) cells were treated with vehicle (0.1% dimethyl sulfoxide) or em08red (10 μM) for 24, 48, and 72 hours and then harvested for fixation. After staining with propidium iodide, cell cycle analysis was carried out using a Canto II cytometer. The data are representative of three independent experiments. (B) Treated FaDu cells were harvested at 12, 24, 36, and 48 hours, and total cell lysates were subjected to Western blot analysis. Antibodies specific for phosphor-Cdk1, total Cdk1, cyclin B, and phosphor-MPM2 were used to detect expression of each protein. Actin expression served as the loading control.
Mentions: A previous study showed that downregulation of ErbB2 protein expression by treatment with neu differential factor could induce arrest of tumor cells in G2/M phase in cells overexpressing ErbB2.21 To determine if em08red has a similar pharmacological effect on cell cycle progression, em08red-treated cells were subjected to flow cytometry analysis. The data indicated significant G2/M accumulation after 24 hours of treatment with em08red in both FaDu and HSC3 cell lines (Figure 3A). G2/M phase-associated proteins, Cdk1 and cyclin B,22 were downregulated after treatment with em08red and phosphor-MPM2 signals were also reduced after em08red, indicating that the treated cells were blocked at G2 phase (Figure 3B). Additionally, we observed a population of sub-G1 em08red-treated HSC3 and FaDu cells after 24 and 48 hours of treatment, respectively, indicative of G2 phase arrest and cell death (Figure 3A). To determine if the molecular mechanisms of em08red-induced cell death occurred via apoptosis or necrosis, the phosphatidylserine level (a marker of apoptosis) on the outer membrane of em08red-treated cells was examined by annexin V staining. As shown in Figure 4A, the percentage annexin V-positive population in em08red-treated FaDu and HSC3 cells increased in a time-dependent manner and correlated with our observations on sub-G1 cell cycle analysis.

Bottom Line: Treatment with em08red significantly downregulated activation of ErbB2 as well as the ErbB2 protein expression level in the tested cell lines and induced G2 arrest.Antiapoptosis protein (Bcl-xl and Bcl-2) expression levels were also downregulated, and active caspase-3 and caspase-9 was detected in cells after treatment with em08red.Moreover, treatment with em08red stimulated production of cytotoxic reactive oxygen species in treated cells, and this could be partially reversed by pretreatment with N-acetylcysteine.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Pharmaceutical Chemistry, China Medical University Hospital, Taichung, Taiwan.

ABSTRACT
Expression of ErbB2 protein is inversely correlated with the prognosis in cancer patients. Consequently, strategies targeting ErbB2 remain an attractive option in treating several types of malignancies, including oral cancer. In addition, many studies have shown that emodin and emodin derivatives are able to inhibit growth of ErbB2-overexpressing tumor cells. In this study, a series of computer modeling-generated emodin analogues were synthesized and tested for their antiproliferative activity against oral cancer cell lines overexpressing ErbB2. Among these analogues, em08red (1,8-dihydroxy-9(10H)-anthracenone) demonstrated potent antiproliferative activity against all three tested ErbB2-overexpressing cell lines, ie, FaDu, HSC3, and OECM1. Treatment with em08red significantly downregulated activation of ErbB2 as well as the ErbB2 protein expression level in the tested cell lines and induced G2 arrest. Antiapoptosis protein (Bcl-xl and Bcl-2) expression levels were also downregulated, and active caspase-3 and caspase-9 was detected in cells after treatment with em08red. Moreover, treatment with em08red stimulated production of cytotoxic reactive oxygen species in treated cells, and this could be partially reversed by pretreatment with N-acetylcysteine. Overall, we demonstrated inhibition of ErbB2 function and induction of reactive oxygen species in tumor cells by em08red, which prevented proliferation of tumor cells and induced apoptotic cell death.

No MeSH data available.


Related in: MedlinePlus