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Curcumin induces apoptosis in gallbladder carcinoma cell line GBC-SD cells.

Liu TY, Tan ZJ, Jiang L, Gu JF, Wu XS, Cao Y, Li ML, Wu KJ, Liu YB - Cancer Cell Int. (2013)

Bottom Line: Morphological changes in apoptotic cells were also found by the Hoechst 33342 staining.After treatment with curcumin fluorescence shifted from red to green as ΔΨm decreased.Taken together, the results indicate that curcumin may be a potential agent for the treatment of gallbladder cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of General Surgery and Department of General Surgery, Xinhua Hospital, Affiliated to Shanghai Jiao Tong University, School of Medicine, No, 1665 Kongjiang Road, Shanghai 200092, China. wukejin_wkj@126.com.

ABSTRACT

Background: Gallbladder carcinoma is a malignant tumor with a very low 5-year survival rate because of the difficulty with its early diagnosis and the very poor prognosis of the advanced cancer state. The aims of this study were to determine whether curcumin could induce the apoptosis of a gallbladder carcinoma cell line, GBC-SD, and to clarify its related mechanism.

Methods: First, the anti-proliferative activities of curcumin-treated and untreated GBC-SD cells were determined using the MTT and colony formation assays. Then, the early apoptosis of cells was detected by the annexin V/propidium iodide double-staining assay and Hoechst 33342 staining assay. Detection of mitochondrial membrane potential was used to validate the ability of curcumin on inducing apoptosis in GBC-SD cells. Cell cycle changes were detected by flow cytometric analysis. Finally, the expressions of the apoptosis-related proteins or genes caspase-3, PARP, Bcl-2, and Bax were analyzed by western blot and quantitative real time PCR assay. Statistical analyses were performed using the Student's t-test for comparison of the results obtained from cells with or without curcumin treatment.

Results: The MTT assay revealed that curcumin had induced a dose- and a time-dependent decrease in cell viability. Colony counting indicated that curcumin had induced a dose-dependent decrease in the colony formation ability in GBC-SD cells. Cells treated with curcumin were arrested at the S phase, according to the flow cytometric analysis. A significant induction of both the early and late phases of apoptosis was shown by the annexin V-FITC and PI staining. Morphological changes in apoptotic cells were also found by the Hoechst 33342 staining. After treatment with curcumin fluorescence shifted from red to green as ΔΨm decreased. Furthermore, western blot and quantitative real time PCR assays demonstrated that the curcumin induced apoptosis in GBC-SD cells by regulating the ratio of Bcl-2/Bax and activating the expression of cleaved caspase-3.

Conclusions: Taken together, the results indicate that curcumin may be a potential agent for the treatment of gallbladder cancer.

No MeSH data available.


Related in: MedlinePlus

Curcumin induces apoptosis in GBC-SD cells. (A) Cells were incubated with curcumin (3.75, 7.5, and 15 μmol/L) for 48 h, followed by staining with annexin-V/PI. The Q3 quadrant (annexin V−/PI−), Q4 quadrant (annexin V+/PI−) and Q2 quadrant (annexin V+/PI+) indicate the percentage of normal cells, early apoptosis and late apoptosis, respectively. (B) Apoptotic nuclear morphology changes induced by curcumin (3.75, 7.5, and 15 μmol/L) treatment for 48 h, were observed by Hoechst 33342 staining in GBC-SD cell lines. (C) Analysis of the mitochondrial membrane potential (ΔΨm). GBC-SD cells were treated with curcumin (3.75, 7.5, and 15 μmol/L) for 48h and then stained with JC-1. Red fluorescence represents mitochondria with intact membrane potential. Green fluorescence represents de-energized mitochondria. Images were taken with a fluorescence microscope.
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Figure 3: Curcumin induces apoptosis in GBC-SD cells. (A) Cells were incubated with curcumin (3.75, 7.5, and 15 μmol/L) for 48 h, followed by staining with annexin-V/PI. The Q3 quadrant (annexin V−/PI−), Q4 quadrant (annexin V+/PI−) and Q2 quadrant (annexin V+/PI+) indicate the percentage of normal cells, early apoptosis and late apoptosis, respectively. (B) Apoptotic nuclear morphology changes induced by curcumin (3.75, 7.5, and 15 μmol/L) treatment for 48 h, were observed by Hoechst 33342 staining in GBC-SD cell lines. (C) Analysis of the mitochondrial membrane potential (ΔΨm). GBC-SD cells were treated with curcumin (3.75, 7.5, and 15 μmol/L) for 48h and then stained with JC-1. Red fluorescence represents mitochondria with intact membrane potential. Green fluorescence represents de-energized mitochondria. Images were taken with a fluorescence microscope.

Mentions: To confirm these results, we evaluated the effects of curcumin on apoptosis in GBC-SD cells by using annexin V-FITC and propidium iodide staining. Normal live cells, phosphatidyl serine (PS) is located on the cytoplasmic surface of the cell membrane. However, in apoptotic cells, PS is translocated from the inner to the outer leaflet of the plasma membrane, thus exposing PS to the external cellular environment. The human anticoagulant, annexin-V, is a 35–36 kDa Ca2+−dependent phospholipid binding protein that has a high affinity for PS. Annexin-V labeled with a fluorophore or biotin can identify apoptotic cells by binding to PS exposed on the outer leaflet.In addition, the red-fluorescent propidium iodide (PI) nucleic acid binding dye is impermeant to live cells and apoptotic cells, but stains dead cells with red fluorescence, binding tightly to the nucleic acids in the cell. After staining a cell population with annexin V and PI, apoptotic cells show green fluorescence, dead cells show red and green fluorescence, and live cells show little or no fluorescence. These populations can easily be distinguished using a flow cytometer. In the scatter plot of double variable flow cytometry, Q3 quadrant(FITC - / PI -) shows living cells; Q2 quadrant (FITC + /PI + ) stands for late apoptotic cells; and Q4 quadrant(FITC + /PI -) represents early apoptotic cells. As assessed by flow cytometry and shown in Figure 3(A), a marked dose-dependent increase in both the early and late stages of apoptosis was obvious in GBC-SD cells after curcumin treatment compared with control cells.


Curcumin induces apoptosis in gallbladder carcinoma cell line GBC-SD cells.

Liu TY, Tan ZJ, Jiang L, Gu JF, Wu XS, Cao Y, Li ML, Wu KJ, Liu YB - Cancer Cell Int. (2013)

Curcumin induces apoptosis in GBC-SD cells. (A) Cells were incubated with curcumin (3.75, 7.5, and 15 μmol/L) for 48 h, followed by staining with annexin-V/PI. The Q3 quadrant (annexin V−/PI−), Q4 quadrant (annexin V+/PI−) and Q2 quadrant (annexin V+/PI+) indicate the percentage of normal cells, early apoptosis and late apoptosis, respectively. (B) Apoptotic nuclear morphology changes induced by curcumin (3.75, 7.5, and 15 μmol/L) treatment for 48 h, were observed by Hoechst 33342 staining in GBC-SD cell lines. (C) Analysis of the mitochondrial membrane potential (ΔΨm). GBC-SD cells were treated with curcumin (3.75, 7.5, and 15 μmol/L) for 48h and then stained with JC-1. Red fluorescence represents mitochondria with intact membrane potential. Green fluorescence represents de-energized mitochondria. Images were taken with a fluorescence microscope.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Curcumin induces apoptosis in GBC-SD cells. (A) Cells were incubated with curcumin (3.75, 7.5, and 15 μmol/L) for 48 h, followed by staining with annexin-V/PI. The Q3 quadrant (annexin V−/PI−), Q4 quadrant (annexin V+/PI−) and Q2 quadrant (annexin V+/PI+) indicate the percentage of normal cells, early apoptosis and late apoptosis, respectively. (B) Apoptotic nuclear morphology changes induced by curcumin (3.75, 7.5, and 15 μmol/L) treatment for 48 h, were observed by Hoechst 33342 staining in GBC-SD cell lines. (C) Analysis of the mitochondrial membrane potential (ΔΨm). GBC-SD cells were treated with curcumin (3.75, 7.5, and 15 μmol/L) for 48h and then stained with JC-1. Red fluorescence represents mitochondria with intact membrane potential. Green fluorescence represents de-energized mitochondria. Images were taken with a fluorescence microscope.
Mentions: To confirm these results, we evaluated the effects of curcumin on apoptosis in GBC-SD cells by using annexin V-FITC and propidium iodide staining. Normal live cells, phosphatidyl serine (PS) is located on the cytoplasmic surface of the cell membrane. However, in apoptotic cells, PS is translocated from the inner to the outer leaflet of the plasma membrane, thus exposing PS to the external cellular environment. The human anticoagulant, annexin-V, is a 35–36 kDa Ca2+−dependent phospholipid binding protein that has a high affinity for PS. Annexin-V labeled with a fluorophore or biotin can identify apoptotic cells by binding to PS exposed on the outer leaflet.In addition, the red-fluorescent propidium iodide (PI) nucleic acid binding dye is impermeant to live cells and apoptotic cells, but stains dead cells with red fluorescence, binding tightly to the nucleic acids in the cell. After staining a cell population with annexin V and PI, apoptotic cells show green fluorescence, dead cells show red and green fluorescence, and live cells show little or no fluorescence. These populations can easily be distinguished using a flow cytometer. In the scatter plot of double variable flow cytometry, Q3 quadrant(FITC - / PI -) shows living cells; Q2 quadrant (FITC + /PI + ) stands for late apoptotic cells; and Q4 quadrant(FITC + /PI -) represents early apoptotic cells. As assessed by flow cytometry and shown in Figure 3(A), a marked dose-dependent increase in both the early and late stages of apoptosis was obvious in GBC-SD cells after curcumin treatment compared with control cells.

Bottom Line: Morphological changes in apoptotic cells were also found by the Hoechst 33342 staining.After treatment with curcumin fluorescence shifted from red to green as ΔΨm decreased.Taken together, the results indicate that curcumin may be a potential agent for the treatment of gallbladder cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of General Surgery and Department of General Surgery, Xinhua Hospital, Affiliated to Shanghai Jiao Tong University, School of Medicine, No, 1665 Kongjiang Road, Shanghai 200092, China. wukejin_wkj@126.com.

ABSTRACT

Background: Gallbladder carcinoma is a malignant tumor with a very low 5-year survival rate because of the difficulty with its early diagnosis and the very poor prognosis of the advanced cancer state. The aims of this study were to determine whether curcumin could induce the apoptosis of a gallbladder carcinoma cell line, GBC-SD, and to clarify its related mechanism.

Methods: First, the anti-proliferative activities of curcumin-treated and untreated GBC-SD cells were determined using the MTT and colony formation assays. Then, the early apoptosis of cells was detected by the annexin V/propidium iodide double-staining assay and Hoechst 33342 staining assay. Detection of mitochondrial membrane potential was used to validate the ability of curcumin on inducing apoptosis in GBC-SD cells. Cell cycle changes were detected by flow cytometric analysis. Finally, the expressions of the apoptosis-related proteins or genes caspase-3, PARP, Bcl-2, and Bax were analyzed by western blot and quantitative real time PCR assay. Statistical analyses were performed using the Student's t-test for comparison of the results obtained from cells with or without curcumin treatment.

Results: The MTT assay revealed that curcumin had induced a dose- and a time-dependent decrease in cell viability. Colony counting indicated that curcumin had induced a dose-dependent decrease in the colony formation ability in GBC-SD cells. Cells treated with curcumin were arrested at the S phase, according to the flow cytometric analysis. A significant induction of both the early and late phases of apoptosis was shown by the annexin V-FITC and PI staining. Morphological changes in apoptotic cells were also found by the Hoechst 33342 staining. After treatment with curcumin fluorescence shifted from red to green as ΔΨm decreased. Furthermore, western blot and quantitative real time PCR assays demonstrated that the curcumin induced apoptosis in GBC-SD cells by regulating the ratio of Bcl-2/Bax and activating the expression of cleaved caspase-3.

Conclusions: Taken together, the results indicate that curcumin may be a potential agent for the treatment of gallbladder cancer.

No MeSH data available.


Related in: MedlinePlus