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Cucurbitacin D induces cell cycle arrest and apoptosis by inhibiting STAT3 and NF-κB signaling in doxorubicin-resistant human breast carcinoma (MCF7/ADR) cells.

Ku JM, Kim SR, Hong SH, Choi HS, Seo HS, Shin YC, Ko SG - Mol. Cell. Biochem. (2015)

Bottom Line: Additionally, cucurbitacin D led to an increase in the IκBα level in the cytosol and a decrease in the p-NF-κB level in the nucleus.Finally, cucurbitacin D inhibited translocation of Stat3 and NF-κB and decreased transcriptional activity in the nucleus.Cucurbitacin D could be used as a useful compound to treat adriamycin-resistant patients.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Clinical Biology and Pharmacogenomics, Department of Preventive Medicine, College of Oriental Medicine, Kyung Hee University, Seoul, 130-701, Republic of Korea.

ABSTRACT
Breast cancer is the most common cancer for women and is a major cause of mortality in women. Doxorubicin is a generally used chemotherapy drug for breast cancer. However, multidrug resistance of breast cancer interferes with the chemotherapy. We examined whether cucurbitacin D affects doxorubicin resistance of MCF7/ADR breast cancer cells. Cell viability was measured by MTT assay. Levels of p-STAT3, p-NF-κB, IκB, and caspases were measured by Western blot analysis. Nuclear staining of Stat3 and NF-κB was measured by immunocytochemistry. STAT3 and NF-κB transcriptional activity was detected by STAT3 and NF-κB luciferase reporter gene assays. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by cucurbitacin D was measured by Annexin V-FITC/propidium iodide assay. More than 90% of MCF7/ADR cells lived upon treatment with doxorubicin for 24 h. However, upon treatment with cucurbitacin D, cell death was more than 60%. Co-administration of cucurbitacin D and doxorubicin induced apoptosis, and G2/M cell cycle arrest, and inhibited upregulated Stat3 by doxorubicin on MCF7/ADR cells. Additionally, cucurbitacin D led to an increase in the IκBα level in the cytosol and a decrease in the p-NF-κB level in the nucleus. Finally, cucurbitacin D inhibited translocation of Stat3 and NF-κB and decreased transcriptional activity in the nucleus. Cucurbitacin D decreases cell proliferation and induces apoptosis by inhibiting Stat3 and NF-κB signaling in doxorubicin-resistant breast cancer cells. Cucurbitacin D could be used as a useful compound to treat adriamycin-resistant patients.

No MeSH data available.


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Cucurbitacin D induces G2/M cell cycle arrest and apoptosis in MCF7/ADR cells. MCF7/ADR cells were treated with cucurbitacin D (0.5 μg/mL) in the presence and absence of doxorubicin (1 μM) for 24 h. Cell cycle distribution was analyzed using a FACS flow cytometer (a). MCF7/ADR cells were treated with the indicated drugs for 24 h and subjected to Annexin V/PI assay. b Effect of cucurbitacin D on the expression of cleaved caspase-3, cleaved caspase-8, and cleaved PARP. MCF7/ADR cells were treated with cucurbitacin D (0.5 μg/mL) in the presence and absence of doxorubicin (1 μM) for 24 h. The cell lysates were subjected to Western blot analysis using specific antibodies (c)
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Fig6: Cucurbitacin D induces G2/M cell cycle arrest and apoptosis in MCF7/ADR cells. MCF7/ADR cells were treated with cucurbitacin D (0.5 μg/mL) in the presence and absence of doxorubicin (1 μM) for 24 h. Cell cycle distribution was analyzed using a FACS flow cytometer (a). MCF7/ADR cells were treated with the indicated drugs for 24 h and subjected to Annexin V/PI assay. b Effect of cucurbitacin D on the expression of cleaved caspase-3, cleaved caspase-8, and cleaved PARP. MCF7/ADR cells were treated with cucurbitacin D (0.5 μg/mL) in the presence and absence of doxorubicin (1 μM) for 24 h. The cell lysates were subjected to Western blot analysis using specific antibodies (c)

Mentions: To investigate whether cucurbitacin D inhibits cell proliferation by promoting changes in cell cycle progression, the effect of cucurbitacin D on the cell cycle profile was assessed using flow cytometry. For this purpose, MCF7/ADR cells were treated with cucurbitacin D (0.5 μg/mL) and/or doxorubicin (1 μM) for 24 h. The results demonstrated that cucurbitacin D induced an increase in the sub-G1 and G2/M populations in MCF7/ADR cells, suggesting that cucurbitacin D induces G2/M cell cycle arrest (Fig. 6a). We found that doxorubicin did not induce apoptosis in drug-resistant cells; however, cucurbitacin D treatment resulted in a 114 % increase in apoptosis (apoptotic cells were calculated after Annexin V/PI staining) when compared to that of control group. Doxorubicin with cucurbitacin D treatment resulted in a 145 % increase in apoptosis (apoptotic cells were calculated after Annexin V/PI staining) when compared to that of doxorubicin control group (Fig. 6b). Because the sub-G1 level was increased by cucurbitacin D treatment, we performed Annexin V/PI staining flow cytometry analysis to detect apoptotic cells. To confirm that caspase activation is involved in cucurbitacin D-induced apoptosis in MCF7/ADR cells, we found that cucurbitacin D up-regulated the levels of cleaved caspase-3, cleaved caspase-8, and cleaved PARP in MCF7/ADR cells (Fig. 6c).Fig. 6


Cucurbitacin D induces cell cycle arrest and apoptosis by inhibiting STAT3 and NF-κB signaling in doxorubicin-resistant human breast carcinoma (MCF7/ADR) cells.

Ku JM, Kim SR, Hong SH, Choi HS, Seo HS, Shin YC, Ko SG - Mol. Cell. Biochem. (2015)

Cucurbitacin D induces G2/M cell cycle arrest and apoptosis in MCF7/ADR cells. MCF7/ADR cells were treated with cucurbitacin D (0.5 μg/mL) in the presence and absence of doxorubicin (1 μM) for 24 h. Cell cycle distribution was analyzed using a FACS flow cytometer (a). MCF7/ADR cells were treated with the indicated drugs for 24 h and subjected to Annexin V/PI assay. b Effect of cucurbitacin D on the expression of cleaved caspase-3, cleaved caspase-8, and cleaved PARP. MCF7/ADR cells were treated with cucurbitacin D (0.5 μg/mL) in the presence and absence of doxorubicin (1 μM) for 24 h. The cell lysates were subjected to Western blot analysis using specific antibodies (c)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig6: Cucurbitacin D induces G2/M cell cycle arrest and apoptosis in MCF7/ADR cells. MCF7/ADR cells were treated with cucurbitacin D (0.5 μg/mL) in the presence and absence of doxorubicin (1 μM) for 24 h. Cell cycle distribution was analyzed using a FACS flow cytometer (a). MCF7/ADR cells were treated with the indicated drugs for 24 h and subjected to Annexin V/PI assay. b Effect of cucurbitacin D on the expression of cleaved caspase-3, cleaved caspase-8, and cleaved PARP. MCF7/ADR cells were treated with cucurbitacin D (0.5 μg/mL) in the presence and absence of doxorubicin (1 μM) for 24 h. The cell lysates were subjected to Western blot analysis using specific antibodies (c)
Mentions: To investigate whether cucurbitacin D inhibits cell proliferation by promoting changes in cell cycle progression, the effect of cucurbitacin D on the cell cycle profile was assessed using flow cytometry. For this purpose, MCF7/ADR cells were treated with cucurbitacin D (0.5 μg/mL) and/or doxorubicin (1 μM) for 24 h. The results demonstrated that cucurbitacin D induced an increase in the sub-G1 and G2/M populations in MCF7/ADR cells, suggesting that cucurbitacin D induces G2/M cell cycle arrest (Fig. 6a). We found that doxorubicin did not induce apoptosis in drug-resistant cells; however, cucurbitacin D treatment resulted in a 114 % increase in apoptosis (apoptotic cells were calculated after Annexin V/PI staining) when compared to that of control group. Doxorubicin with cucurbitacin D treatment resulted in a 145 % increase in apoptosis (apoptotic cells were calculated after Annexin V/PI staining) when compared to that of doxorubicin control group (Fig. 6b). Because the sub-G1 level was increased by cucurbitacin D treatment, we performed Annexin V/PI staining flow cytometry analysis to detect apoptotic cells. To confirm that caspase activation is involved in cucurbitacin D-induced apoptosis in MCF7/ADR cells, we found that cucurbitacin D up-regulated the levels of cleaved caspase-3, cleaved caspase-8, and cleaved PARP in MCF7/ADR cells (Fig. 6c).Fig. 6

Bottom Line: Additionally, cucurbitacin D led to an increase in the IκBα level in the cytosol and a decrease in the p-NF-κB level in the nucleus.Finally, cucurbitacin D inhibited translocation of Stat3 and NF-κB and decreased transcriptional activity in the nucleus.Cucurbitacin D could be used as a useful compound to treat adriamycin-resistant patients.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Clinical Biology and Pharmacogenomics, Department of Preventive Medicine, College of Oriental Medicine, Kyung Hee University, Seoul, 130-701, Republic of Korea.

ABSTRACT
Breast cancer is the most common cancer for women and is a major cause of mortality in women. Doxorubicin is a generally used chemotherapy drug for breast cancer. However, multidrug resistance of breast cancer interferes with the chemotherapy. We examined whether cucurbitacin D affects doxorubicin resistance of MCF7/ADR breast cancer cells. Cell viability was measured by MTT assay. Levels of p-STAT3, p-NF-κB, IκB, and caspases were measured by Western blot analysis. Nuclear staining of Stat3 and NF-κB was measured by immunocytochemistry. STAT3 and NF-κB transcriptional activity was detected by STAT3 and NF-κB luciferase reporter gene assays. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by cucurbitacin D was measured by Annexin V-FITC/propidium iodide assay. More than 90% of MCF7/ADR cells lived upon treatment with doxorubicin for 24 h. However, upon treatment with cucurbitacin D, cell death was more than 60%. Co-administration of cucurbitacin D and doxorubicin induced apoptosis, and G2/M cell cycle arrest, and inhibited upregulated Stat3 by doxorubicin on MCF7/ADR cells. Additionally, cucurbitacin D led to an increase in the IκBα level in the cytosol and a decrease in the p-NF-κB level in the nucleus. Finally, cucurbitacin D inhibited translocation of Stat3 and NF-κB and decreased transcriptional activity in the nucleus. Cucurbitacin D decreases cell proliferation and induces apoptosis by inhibiting Stat3 and NF-κB signaling in doxorubicin-resistant breast cancer cells. Cucurbitacin D could be used as a useful compound to treat adriamycin-resistant patients.

No MeSH data available.


Related in: MedlinePlus