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Apoptosis Induction in Primary Human Colorectal Cancer Cell Lines and Retarded Tumor Growth in SCID Mice by Sulforaphane.

Chen MJ, Tang WY, Hsu CW, Tsai YT, Wu JF, Lin CW, Cheng YM, Hsu YC - Evid Based Complement Alternat Med (2011)

Bottom Line: In addition, treated cells showed nuclear apoptotic morphology that coincided with an activation of caspase-3, and loss of mitochondrial membrane potential (ΔΨm).Incubations at higher SFN doses (12.5 and 25 μM) resulted in cleavage of procaspase-3 and elevated caspase-2, -3, -8, and -9 activity, suggesting that the induction of apoptosis and the sulforaphane-induced mitosis delay at the lower dose are independently regulated.Daily SFN s.c. injections (400 micromol/kg/d for 3 weeks) in severe combined immunodeficient mice with primary human CRC (CP1 to CP5) s.c. tumors resulted in a decrease of mean tumor weight by 70% compared with vehicle-treated controls.

View Article: PubMed Central - PubMed

Affiliation: Division of Traumatology, Department of Surgery, Chi Mei Medical Center, Tainan, Taiwan.

ABSTRACT
We have investigated the anticancer effects of the dietary isothiocyanate sulforaphane (SFN) on colorectal cancer (CRC), using primary cancer cells lines isolated from five Taiwanese colorectal cancer patients as the model for colorectal cancer. SFN-treated cells accumulated in metaphase (SFN 6.25 μM) and subG1 (SFN 12.5 and 25 μM) as determined by flow cytometry. In addition, treated cells showed nuclear apoptotic morphology that coincided with an activation of caspase-3, and loss of mitochondrial membrane potential (ΔΨm). Incubations at higher SFN doses (12.5 and 25 μM) resulted in cleavage of procaspase-3 and elevated caspase-2, -3, -8, and -9 activity, suggesting that the induction of apoptosis and the sulforaphane-induced mitosis delay at the lower dose are independently regulated. Daily SFN s.c. injections (400 micromol/kg/d for 3 weeks) in severe combined immunodeficient mice with primary human CRC (CP1 to CP5) s.c. tumors resulted in a decrease of mean tumor weight by 70% compared with vehicle-treated controls. Our findings suggest that, in addition to the known effects on cancer prevention, sulforaphane may have antitumor activity in established colorectal cancer.

No MeSH data available.


Related in: MedlinePlus

Reduction of the mitochondrial membrane potential (ΔΨm) in the CRC cell lines by SFN, which was determined by JC-1 staining and detected by the flow cytometry. (a) Dot plots of five CRC cell lines with SFN treatment at 0, 6.25, 12.5, and 25 μM for 6 h. (b) The reduction of the ΔΨm containing polarized or depolarized mitochondria determined the ratio of the two fluorescence intensities analyzed by flow cytometry. All the data shown are the folds of five CRC cell lines. The symbol (∗) on each group of bars denotes that difference from the treatment with 0 μM SFN is statistically significant at P < 0.05.
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fig4: Reduction of the mitochondrial membrane potential (ΔΨm) in the CRC cell lines by SFN, which was determined by JC-1 staining and detected by the flow cytometry. (a) Dot plots of five CRC cell lines with SFN treatment at 0, 6.25, 12.5, and 25 μM for 6 h. (b) The reduction of the ΔΨm containing polarized or depolarized mitochondria determined the ratio of the two fluorescence intensities analyzed by flow cytometry. All the data shown are the folds of five CRC cell lines. The symbol (∗) on each group of bars denotes that difference from the treatment with 0 μM SFN is statistically significant at P < 0.05.

Mentions: The loss of mitochondrial membrane potential is a hallmark for apoptosis. It is an early event coinciding with caspase activation. In nonapoptotic cells, JC-1 exists as a monomer in the cytosol (green) and accumulates as aggregates in the mitochondria, which appear red. In apoptotic and necrotic cells, JC-1 exists in monomeric form and stains the cytosol green. Figure 4(a) shows typical FL-1/FL-2 dot plots for JC-1 staining CRC cell lines with and without apoptosis. SFN-free CRC cell lines are without apoptosis, which have red fluorescing J-aggregates. The green fluorescing monomers shown in the lower part indicate apoptotic cell lines (SFN 6.25, 12.5, and 25 μM treatment). Figure 4(b) shows the percentages of apoptotic CRC cell lines analyzed by flow cytometer in different SFN-treated groups. The x-fold increase of mitochondrial membrane potential lost was observed in all the doses after treatment for 6 h. In 6 h, the folds increased to 1.8 + 0.6 with 6.25 μM SFN treatment. When the concentrations of SFN increased to 12.5 and 25 μM, the folds raised to 2.3 + 0.9 and 3.5 + 0.5, respectively. Taken together, the observations imply that SFN has significantly reduced the mitochondrial membrane potential of CRC cell lines.


Apoptosis Induction in Primary Human Colorectal Cancer Cell Lines and Retarded Tumor Growth in SCID Mice by Sulforaphane.

Chen MJ, Tang WY, Hsu CW, Tsai YT, Wu JF, Lin CW, Cheng YM, Hsu YC - Evid Based Complement Alternat Med (2011)

Reduction of the mitochondrial membrane potential (ΔΨm) in the CRC cell lines by SFN, which was determined by JC-1 staining and detected by the flow cytometry. (a) Dot plots of five CRC cell lines with SFN treatment at 0, 6.25, 12.5, and 25 μM for 6 h. (b) The reduction of the ΔΨm containing polarized or depolarized mitochondria determined the ratio of the two fluorescence intensities analyzed by flow cytometry. All the data shown are the folds of five CRC cell lines. The symbol (∗) on each group of bars denotes that difference from the treatment with 0 μM SFN is statistically significant at P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139908&req=5

fig4: Reduction of the mitochondrial membrane potential (ΔΨm) in the CRC cell lines by SFN, which was determined by JC-1 staining and detected by the flow cytometry. (a) Dot plots of five CRC cell lines with SFN treatment at 0, 6.25, 12.5, and 25 μM for 6 h. (b) The reduction of the ΔΨm containing polarized or depolarized mitochondria determined the ratio of the two fluorescence intensities analyzed by flow cytometry. All the data shown are the folds of five CRC cell lines. The symbol (∗) on each group of bars denotes that difference from the treatment with 0 μM SFN is statistically significant at P < 0.05.
Mentions: The loss of mitochondrial membrane potential is a hallmark for apoptosis. It is an early event coinciding with caspase activation. In nonapoptotic cells, JC-1 exists as a monomer in the cytosol (green) and accumulates as aggregates in the mitochondria, which appear red. In apoptotic and necrotic cells, JC-1 exists in monomeric form and stains the cytosol green. Figure 4(a) shows typical FL-1/FL-2 dot plots for JC-1 staining CRC cell lines with and without apoptosis. SFN-free CRC cell lines are without apoptosis, which have red fluorescing J-aggregates. The green fluorescing monomers shown in the lower part indicate apoptotic cell lines (SFN 6.25, 12.5, and 25 μM treatment). Figure 4(b) shows the percentages of apoptotic CRC cell lines analyzed by flow cytometer in different SFN-treated groups. The x-fold increase of mitochondrial membrane potential lost was observed in all the doses after treatment for 6 h. In 6 h, the folds increased to 1.8 + 0.6 with 6.25 μM SFN treatment. When the concentrations of SFN increased to 12.5 and 25 μM, the folds raised to 2.3 + 0.9 and 3.5 + 0.5, respectively. Taken together, the observations imply that SFN has significantly reduced the mitochondrial membrane potential of CRC cell lines.

Bottom Line: In addition, treated cells showed nuclear apoptotic morphology that coincided with an activation of caspase-3, and loss of mitochondrial membrane potential (ΔΨm).Incubations at higher SFN doses (12.5 and 25 μM) resulted in cleavage of procaspase-3 and elevated caspase-2, -3, -8, and -9 activity, suggesting that the induction of apoptosis and the sulforaphane-induced mitosis delay at the lower dose are independently regulated.Daily SFN s.c. injections (400 micromol/kg/d for 3 weeks) in severe combined immunodeficient mice with primary human CRC (CP1 to CP5) s.c. tumors resulted in a decrease of mean tumor weight by 70% compared with vehicle-treated controls.

View Article: PubMed Central - PubMed

Affiliation: Division of Traumatology, Department of Surgery, Chi Mei Medical Center, Tainan, Taiwan.

ABSTRACT
We have investigated the anticancer effects of the dietary isothiocyanate sulforaphane (SFN) on colorectal cancer (CRC), using primary cancer cells lines isolated from five Taiwanese colorectal cancer patients as the model for colorectal cancer. SFN-treated cells accumulated in metaphase (SFN 6.25 μM) and subG1 (SFN 12.5 and 25 μM) as determined by flow cytometry. In addition, treated cells showed nuclear apoptotic morphology that coincided with an activation of caspase-3, and loss of mitochondrial membrane potential (ΔΨm). Incubations at higher SFN doses (12.5 and 25 μM) resulted in cleavage of procaspase-3 and elevated caspase-2, -3, -8, and -9 activity, suggesting that the induction of apoptosis and the sulforaphane-induced mitosis delay at the lower dose are independently regulated. Daily SFN s.c. injections (400 micromol/kg/d for 3 weeks) in severe combined immunodeficient mice with primary human CRC (CP1 to CP5) s.c. tumors resulted in a decrease of mean tumor weight by 70% compared with vehicle-treated controls. Our findings suggest that, in addition to the known effects on cancer prevention, sulforaphane may have antitumor activity in established colorectal cancer.

No MeSH data available.


Related in: MedlinePlus