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Antimicrobial peptide FF/CAP18 induces apoptotic cell death in HCT116 colon cancer cells via changes in the metabolic profile.

Kuroda K, Fukuda T, Isogai H, Okumura K, Krstic-Demonacos M, Isogai E - Int. J. Oncol. (2015)

Bottom Line: We previously reported that cathelicidin-related or modified antimicrobial peptides, such as FF/CAP18, have antiproliferative effects on the squamous cell carcinoma cell line SAS-H1, and the colon carcinoma cell line HCT116.Purine metabolism, glycolysis, and the TCA cycle, were altered in FF/CAP18-treated cells in a dose-dependent manner.Our present study provides mechanistic insights into the anticancer effects of antimicrobial peptides that show great potential as new therapies for colon cancer.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan.

ABSTRACT
Metabolic reprogramming is one of the hallmarks of cancer and can be targeted by therapeutic agents. We previously reported that cathelicidin-related or modified antimicrobial peptides, such as FF/CAP18, have antiproliferative effects on the squamous cell carcinoma cell line SAS-H1, and the colon carcinoma cell line HCT116. Although antimicrobial peptides have potential use in the development of new therapeutic strategies, their effects on the metabolism of cancer cells are poorly understood. Here, we investigated changes in the levels of metabolites in HCT116 cells caused by FF/CAP18, via capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Analysis of the 177 intracellular metabolites and 113 metabolites in conditioned medium that were detected by CE-TOFMS, revealed dramatic changes in the metabolic profile of HCT116 cells after treatment with FF/CAP18. The metabolic profile showed that the levels of most metabolites in the major metabolic pathways supported the rapid proliferation of cancer cells. Purine metabolism, glycolysis, and the TCA cycle, were altered in FF/CAP18-treated cells in a dose-dependent manner. Our present study provides mechanistic insights into the anticancer effects of antimicrobial peptides that show great potential as new therapies for colon cancer.

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Detection of apoptosis of HCT116 cells after treatment with FF/CAP18. Cells were treated with 10 or 40 μg/ml FF/CAP18 for 96 h and subjected to the combined Annexin V binding-7-AAD staining assay. (A) Representative results of the assay carried out with non-treated HCT116 cells (left panel), and with HCT116 cells treated with FF/CAP18 at 10 μg/ml (middle panel) or 40 μg/ml (right panel). Based on the reactivity with Annexin V and the intensity of the 7-AAD fluorescence, cells can be classified into four categories: dead, live, early apoptosis and late apoptosis/dead. Triplicate experiments were conducted and representative results are shown. (B) The percentage of live cells (left panel), cells in early apoptosis (middle panel) and cells in late apoptosis or dead (right panel). Triplicate samples were used to obtain the mean and standard deviation. The asterisks indicate statistical significance. *P<0.05.
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f1-ijo-46-04-1516: Detection of apoptosis of HCT116 cells after treatment with FF/CAP18. Cells were treated with 10 or 40 μg/ml FF/CAP18 for 96 h and subjected to the combined Annexin V binding-7-AAD staining assay. (A) Representative results of the assay carried out with non-treated HCT116 cells (left panel), and with HCT116 cells treated with FF/CAP18 at 10 μg/ml (middle panel) or 40 μg/ml (right panel). Based on the reactivity with Annexin V and the intensity of the 7-AAD fluorescence, cells can be classified into four categories: dead, live, early apoptosis and late apoptosis/dead. Triplicate experiments were conducted and representative results are shown. (B) The percentage of live cells (left panel), cells in early apoptosis (middle panel) and cells in late apoptosis or dead (right panel). Triplicate samples were used to obtain the mean and standard deviation. The asterisks indicate statistical significance. *P<0.05.

Mentions: Combined Annexin V and 7-AAD reactivity allowed classification of cells into four groups, as follows: early apoptotic cells [Annexin V (+) and 7-AAD (−)], late apoptotic or dead cells [Annexin V (+) and 7-AAD (+)], dead cells [Annexin V (−) and 7-AAD (+)], and live cells [Annexin V (−) and 7-AAD (−)]; see the scatter plots in Fig. 1A. Treatment of HCT116 cells with FF/CAP18 at 10 μg/ml induced high affinity for Annexin V, as shown by the right shift of the scatter plot compared with that of non-treated cells, indicating early apoptosis (Fig. 1A, middle panel). On the other hand, FF/CAP18 treatment at 40 μg/ml increased the number of cells that were positive for Annexin V (+) and 7-AAD (+), indicating that a high dose of FF/CAP18 induced apoptotic cell death in HCT116 cells. The ratio of HCT116 cells at each stage of apoptosis after treatment with the two different doses of FF/CAP18 is summarized in Fig. 1B. The percentage of live cells decreased significantly in a dose-dependent manner (Fig. 1B). The percentage of cells in early apoptosis, however, significantly increased with 10 μg/ml FF/CAP18 treatment, whereas, the percentage of late apoptotic and dead cells only increased with 40 μg/ml treatment (Fig. 1B). From these results, we concluded that early-stage apoptosis was induced by a comparatively low dose (10 μg/ml) of FF/CAP18, whereas high-dose treatment (40 μg/ml) could cause late-stage apoptosis and cell death.


Antimicrobial peptide FF/CAP18 induces apoptotic cell death in HCT116 colon cancer cells via changes in the metabolic profile.

Kuroda K, Fukuda T, Isogai H, Okumura K, Krstic-Demonacos M, Isogai E - Int. J. Oncol. (2015)

Detection of apoptosis of HCT116 cells after treatment with FF/CAP18. Cells were treated with 10 or 40 μg/ml FF/CAP18 for 96 h and subjected to the combined Annexin V binding-7-AAD staining assay. (A) Representative results of the assay carried out with non-treated HCT116 cells (left panel), and with HCT116 cells treated with FF/CAP18 at 10 μg/ml (middle panel) or 40 μg/ml (right panel). Based on the reactivity with Annexin V and the intensity of the 7-AAD fluorescence, cells can be classified into four categories: dead, live, early apoptosis and late apoptosis/dead. Triplicate experiments were conducted and representative results are shown. (B) The percentage of live cells (left panel), cells in early apoptosis (middle panel) and cells in late apoptosis or dead (right panel). Triplicate samples were used to obtain the mean and standard deviation. The asterisks indicate statistical significance. *P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1-ijo-46-04-1516: Detection of apoptosis of HCT116 cells after treatment with FF/CAP18. Cells were treated with 10 or 40 μg/ml FF/CAP18 for 96 h and subjected to the combined Annexin V binding-7-AAD staining assay. (A) Representative results of the assay carried out with non-treated HCT116 cells (left panel), and with HCT116 cells treated with FF/CAP18 at 10 μg/ml (middle panel) or 40 μg/ml (right panel). Based on the reactivity with Annexin V and the intensity of the 7-AAD fluorescence, cells can be classified into four categories: dead, live, early apoptosis and late apoptosis/dead. Triplicate experiments were conducted and representative results are shown. (B) The percentage of live cells (left panel), cells in early apoptosis (middle panel) and cells in late apoptosis or dead (right panel). Triplicate samples were used to obtain the mean and standard deviation. The asterisks indicate statistical significance. *P<0.05.
Mentions: Combined Annexin V and 7-AAD reactivity allowed classification of cells into four groups, as follows: early apoptotic cells [Annexin V (+) and 7-AAD (−)], late apoptotic or dead cells [Annexin V (+) and 7-AAD (+)], dead cells [Annexin V (−) and 7-AAD (+)], and live cells [Annexin V (−) and 7-AAD (−)]; see the scatter plots in Fig. 1A. Treatment of HCT116 cells with FF/CAP18 at 10 μg/ml induced high affinity for Annexin V, as shown by the right shift of the scatter plot compared with that of non-treated cells, indicating early apoptosis (Fig. 1A, middle panel). On the other hand, FF/CAP18 treatment at 40 μg/ml increased the number of cells that were positive for Annexin V (+) and 7-AAD (+), indicating that a high dose of FF/CAP18 induced apoptotic cell death in HCT116 cells. The ratio of HCT116 cells at each stage of apoptosis after treatment with the two different doses of FF/CAP18 is summarized in Fig. 1B. The percentage of live cells decreased significantly in a dose-dependent manner (Fig. 1B). The percentage of cells in early apoptosis, however, significantly increased with 10 μg/ml FF/CAP18 treatment, whereas, the percentage of late apoptotic and dead cells only increased with 40 μg/ml treatment (Fig. 1B). From these results, we concluded that early-stage apoptosis was induced by a comparatively low dose (10 μg/ml) of FF/CAP18, whereas high-dose treatment (40 μg/ml) could cause late-stage apoptosis and cell death.

Bottom Line: We previously reported that cathelicidin-related or modified antimicrobial peptides, such as FF/CAP18, have antiproliferative effects on the squamous cell carcinoma cell line SAS-H1, and the colon carcinoma cell line HCT116.Purine metabolism, glycolysis, and the TCA cycle, were altered in FF/CAP18-treated cells in a dose-dependent manner.Our present study provides mechanistic insights into the anticancer effects of antimicrobial peptides that show great potential as new therapies for colon cancer.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan.

ABSTRACT
Metabolic reprogramming is one of the hallmarks of cancer and can be targeted by therapeutic agents. We previously reported that cathelicidin-related or modified antimicrobial peptides, such as FF/CAP18, have antiproliferative effects on the squamous cell carcinoma cell line SAS-H1, and the colon carcinoma cell line HCT116. Although antimicrobial peptides have potential use in the development of new therapeutic strategies, their effects on the metabolism of cancer cells are poorly understood. Here, we investigated changes in the levels of metabolites in HCT116 cells caused by FF/CAP18, via capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Analysis of the 177 intracellular metabolites and 113 metabolites in conditioned medium that were detected by CE-TOFMS, revealed dramatic changes in the metabolic profile of HCT116 cells after treatment with FF/CAP18. The metabolic profile showed that the levels of most metabolites in the major metabolic pathways supported the rapid proliferation of cancer cells. Purine metabolism, glycolysis, and the TCA cycle, were altered in FF/CAP18-treated cells in a dose-dependent manner. Our present study provides mechanistic insights into the anticancer effects of antimicrobial peptides that show great potential as new therapies for colon cancer.

Show MeSH
Related in: MedlinePlus