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An evaluation of the anti-angiogenic effect of the Korean medicinal formula "Sa-mi-yeon-geon-tang" in vitro and in ovo.

Yi JM, Bang OS, Kim NS - BMC Complement Altern Med (2015)

Bottom Line: These anti-angiogenic effects of SMYGT in HUVECs were related to decreases in the phosphorylation of focal adhesion kinase and the expression of matrix metallopeptidase-2 activity.SMYGT exhibited an anti-angiogenic potential in both in vitro and in ovo experiments, which may partially contribute to its anti-tumor effect in clinical conditions.We suggest that SMYGT may be a promising source material for the development of anti-cancer chemotherapeutics that target angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: KM-Based Herbal Drug Development Group, Korea Institute of Oriental Medicine, 305-811, Daejeon, Republic of Korea. jmyi@kiom.re.kr.

ABSTRACT

Background: Angiogenesis is a general hallmark of cancer; therefore, the inhibition of tumor-derived angiogenesis is considered to be an attractive target in the development of anti-cancer agents. Sa-mi-yeon-geon-tang (SMYGT), a decoction that consists of four natural medicinal products, has been traditionally prescribed in Oriental medicine to treat diverse diseases, including cancer. In the present study, we investigated the anti-angiogenic potential of SMYGT in vitro and in ovo.

Methods: The anti-angiogenic potential of SMYGT was evaluated using conventional in vitro assays with human umbilical vein endothelial cells (HUVECs) and chorioallantoic membrane (CAM) assays with fertilized eggs. The expression changes of pro-angiogenic proteins and intracellular signaling in HUVECs following SMYGT treatment were determined by quantitative polymerase chain reaction, gelatinase zymography, and western blot analysis.

Results: SMYGT efficiently inhibited three-dimensional capillary-like tube formation by HUVECs on extracellular matrix supports, as well as new vessel formation on CAMs. SMYGT inhibited cell adhesion to the extracellular matrix and HUVEC cell invasion through Matrigel without affecting cell proliferation, viability, and motility. These anti-angiogenic effects of SMYGT in HUVECs were related to decreases in the phosphorylation of focal adhesion kinase and the expression of matrix metallopeptidase-2 activity.

Conclusions: SMYGT exhibited an anti-angiogenic potential in both in vitro and in ovo experiments, which may partially contribute to its anti-tumor effect in clinical conditions. We suggest that SMYGT may be a promising source material for the development of anti-cancer chemotherapeutics that target angiogenesis.

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Effects of SMYGT on the HUVEC-mediated angiogenic process in vitro. (A) HUVECs were cultured in the presence and absence of SMYGT for 24 h and measured using an automatic cell counter to determine cell growth and viability. (B) Scratches were applied to the lawn of HUVECs, and then cells were cultured in the presence or absence of SMYGT. After 12 h, the relative cell motility was determined by analyzing digital images. (C) HUVECs were resuspended in culture medium containing various concentrations of SMYGT and then inoculated in culture plates that were pre-coated with Matrigel. After 2 h, the adherent cells were fixed, stained, and then counted under a microscope. (D) Serum-starved HUVECs were inoculated into the cell culture inserts pre-coated with Matrigel and then cultured in the presence or absence of SMYGT. After 16 h, chemotactic HUVECs that invaded through the Matrigel were stained and counted under a microscope. Sulforaphane (Sulfo) was included in these assays for a positive control drug. All data, except viability, are presented as the relative means ± S.D. of triplicate experiments compared to the vehicle treatment group. *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig2: Effects of SMYGT on the HUVEC-mediated angiogenic process in vitro. (A) HUVECs were cultured in the presence and absence of SMYGT for 24 h and measured using an automatic cell counter to determine cell growth and viability. (B) Scratches were applied to the lawn of HUVECs, and then cells were cultured in the presence or absence of SMYGT. After 12 h, the relative cell motility was determined by analyzing digital images. (C) HUVECs were resuspended in culture medium containing various concentrations of SMYGT and then inoculated in culture plates that were pre-coated with Matrigel. After 2 h, the adherent cells were fixed, stained, and then counted under a microscope. (D) Serum-starved HUVECs were inoculated into the cell culture inserts pre-coated with Matrigel and then cultured in the presence or absence of SMYGT. After 16 h, chemotactic HUVECs that invaded through the Matrigel were stained and counted under a microscope. Sulforaphane (Sulfo) was included in these assays for a positive control drug. All data, except viability, are presented as the relative means ± S.D. of triplicate experiments compared to the vehicle treatment group. *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: To understand the anti-angiogenic activity of SMYGT, the cellular responses of HUVECs to SMYGT treatment were investigated in vitro. First, we determined the cytotoxicity of SMYGT in HUVECs based on cellular membrane integrity. When HUVECs were exposed to increasing concentrations of SMYGT, no significant morphological changes was observed (Additional file 1B), and HUVECs maintained normal cell growth and a viability of > 90% in concentrations up to 400 μg/mL of SMYGT (Figure 2A). These data suggest that the anti-angiogenic potential of SMYGT, as demonstrated by HUVEC-mediated tube formation and in ovo CAM assays, was not due to its cytotoxicity. An in vitro wound healing assay used to determine cell mobility demonstrated that although a slight inhibitory effect was observed at SMYGT concentrations ≥200 μg/mL, but HUVECs showed normal cell movement when compared to cells treated with 5 μM of sulforaphane, a positive control drug (Additional file 1C). Image analysis did not reveal that SMYGT induced any significant changes in cell movement (Figure 2B). In contrast, SMYGT reduced endothelial cell adhesion to the extracellular matrix (Additional file 1D), and image analysis revealed that 400 μg/mL of SMYGT reduced cell adhesion by 80.6% (Figure 2C). Furthermore, SMYGT reduced cellular invasiveness through the membrane (Additional file 1E), and the inhibitory effect of 200 μg/mL of SMYGT was comparable to that of 5 μM of sulforaphane (Figure 2D).Figure 2


An evaluation of the anti-angiogenic effect of the Korean medicinal formula "Sa-mi-yeon-geon-tang" in vitro and in ovo.

Yi JM, Bang OS, Kim NS - BMC Complement Altern Med (2015)

Effects of SMYGT on the HUVEC-mediated angiogenic process in vitro. (A) HUVECs were cultured in the presence and absence of SMYGT for 24 h and measured using an automatic cell counter to determine cell growth and viability. (B) Scratches were applied to the lawn of HUVECs, and then cells were cultured in the presence or absence of SMYGT. After 12 h, the relative cell motility was determined by analyzing digital images. (C) HUVECs were resuspended in culture medium containing various concentrations of SMYGT and then inoculated in culture plates that were pre-coated with Matrigel. After 2 h, the adherent cells were fixed, stained, and then counted under a microscope. (D) Serum-starved HUVECs were inoculated into the cell culture inserts pre-coated with Matrigel and then cultured in the presence or absence of SMYGT. After 16 h, chemotactic HUVECs that invaded through the Matrigel were stained and counted under a microscope. Sulforaphane (Sulfo) was included in these assays for a positive control drug. All data, except viability, are presented as the relative means ± S.D. of triplicate experiments compared to the vehicle treatment group. *P < 0.05, **P < 0.01, ***P < 0.001.
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Related In: Results  -  Collection

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Fig2: Effects of SMYGT on the HUVEC-mediated angiogenic process in vitro. (A) HUVECs were cultured in the presence and absence of SMYGT for 24 h and measured using an automatic cell counter to determine cell growth and viability. (B) Scratches were applied to the lawn of HUVECs, and then cells were cultured in the presence or absence of SMYGT. After 12 h, the relative cell motility was determined by analyzing digital images. (C) HUVECs were resuspended in culture medium containing various concentrations of SMYGT and then inoculated in culture plates that were pre-coated with Matrigel. After 2 h, the adherent cells were fixed, stained, and then counted under a microscope. (D) Serum-starved HUVECs were inoculated into the cell culture inserts pre-coated with Matrigel and then cultured in the presence or absence of SMYGT. After 16 h, chemotactic HUVECs that invaded through the Matrigel were stained and counted under a microscope. Sulforaphane (Sulfo) was included in these assays for a positive control drug. All data, except viability, are presented as the relative means ± S.D. of triplicate experiments compared to the vehicle treatment group. *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: To understand the anti-angiogenic activity of SMYGT, the cellular responses of HUVECs to SMYGT treatment were investigated in vitro. First, we determined the cytotoxicity of SMYGT in HUVECs based on cellular membrane integrity. When HUVECs were exposed to increasing concentrations of SMYGT, no significant morphological changes was observed (Additional file 1B), and HUVECs maintained normal cell growth and a viability of > 90% in concentrations up to 400 μg/mL of SMYGT (Figure 2A). These data suggest that the anti-angiogenic potential of SMYGT, as demonstrated by HUVEC-mediated tube formation and in ovo CAM assays, was not due to its cytotoxicity. An in vitro wound healing assay used to determine cell mobility demonstrated that although a slight inhibitory effect was observed at SMYGT concentrations ≥200 μg/mL, but HUVECs showed normal cell movement when compared to cells treated with 5 μM of sulforaphane, a positive control drug (Additional file 1C). Image analysis did not reveal that SMYGT induced any significant changes in cell movement (Figure 2B). In contrast, SMYGT reduced endothelial cell adhesion to the extracellular matrix (Additional file 1D), and image analysis revealed that 400 μg/mL of SMYGT reduced cell adhesion by 80.6% (Figure 2C). Furthermore, SMYGT reduced cellular invasiveness through the membrane (Additional file 1E), and the inhibitory effect of 200 μg/mL of SMYGT was comparable to that of 5 μM of sulforaphane (Figure 2D).Figure 2

Bottom Line: These anti-angiogenic effects of SMYGT in HUVECs were related to decreases in the phosphorylation of focal adhesion kinase and the expression of matrix metallopeptidase-2 activity.SMYGT exhibited an anti-angiogenic potential in both in vitro and in ovo experiments, which may partially contribute to its anti-tumor effect in clinical conditions.We suggest that SMYGT may be a promising source material for the development of anti-cancer chemotherapeutics that target angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: KM-Based Herbal Drug Development Group, Korea Institute of Oriental Medicine, 305-811, Daejeon, Republic of Korea. jmyi@kiom.re.kr.

ABSTRACT

Background: Angiogenesis is a general hallmark of cancer; therefore, the inhibition of tumor-derived angiogenesis is considered to be an attractive target in the development of anti-cancer agents. Sa-mi-yeon-geon-tang (SMYGT), a decoction that consists of four natural medicinal products, has been traditionally prescribed in Oriental medicine to treat diverse diseases, including cancer. In the present study, we investigated the anti-angiogenic potential of SMYGT in vitro and in ovo.

Methods: The anti-angiogenic potential of SMYGT was evaluated using conventional in vitro assays with human umbilical vein endothelial cells (HUVECs) and chorioallantoic membrane (CAM) assays with fertilized eggs. The expression changes of pro-angiogenic proteins and intracellular signaling in HUVECs following SMYGT treatment were determined by quantitative polymerase chain reaction, gelatinase zymography, and western blot analysis.

Results: SMYGT efficiently inhibited three-dimensional capillary-like tube formation by HUVECs on extracellular matrix supports, as well as new vessel formation on CAMs. SMYGT inhibited cell adhesion to the extracellular matrix and HUVEC cell invasion through Matrigel without affecting cell proliferation, viability, and motility. These anti-angiogenic effects of SMYGT in HUVECs were related to decreases in the phosphorylation of focal adhesion kinase and the expression of matrix metallopeptidase-2 activity.

Conclusions: SMYGT exhibited an anti-angiogenic potential in both in vitro and in ovo experiments, which may partially contribute to its anti-tumor effect in clinical conditions. We suggest that SMYGT may be a promising source material for the development of anti-cancer chemotherapeutics that target angiogenesis.

Show MeSH
Related in: MedlinePlus