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Molecular insights into the anti-cancer properties of traditional Tibetan medicine Yukyung Karne.

Choedon T, Dolma D, Mathan G, Kumar V - BMC Complement Altern Med (2014)

Bottom Line: Confocal microscopy suggested inhibition of cell proliferation and increase in cytochrome c release via perturbation in mitochondrial membrane potential (Δψm).Further, YK up-regulated the expression of tumor suppressor p53 and key cyclin-dependent kinase inhibitor p21, and inhibited VEGF secretion by cells.Interestingly, YK also exhibited a synergy with paclitaxel which is a well-known anti-cancer therapeutic drug.

View Article: PubMed Central - PubMed

Affiliation: Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. vijay@icgeb.res.in.

ABSTRACT

Background: Yukyung karne (YK) is a traditional Tibetan formulation used for many centuries for the treatment of ovarian cancer. However, the pharmacological basis of its anticancer property is not well understood. In the present study, the anticancer property of YK was investigated in cell culture.

Methods: The growth inhibitory property of YK was evaluated in SKOV6, IHH, HepG2 and HEK293 cell lines using MTT assay. The pro-apoptotic activity of drug was analyzed by terminal deoxynuleotidyl transferase dUTP nick end labeling (TUNEL) and DNA fragmentation assays. Confocal microscopy was used to show the release of cytochrome c and its co-localization with mitochondria with the help of dsRed mitotracker in SKOV6 cells. The inhibition in cell proliferation was also visualized by confocal microscopy after BrDU incorporation. The activation of tumor suppressor p53 was evaluated by Western blotting while VEGF levels in culture supernatant were measured by a colorimetric method.

Results: YK specifically and efficiently induced apoptotic killing of the human ovarian cancer SKOV6 cells as indicated by increased DNA fragmentation and nick end DNA labeling. Confocal microscopy suggested inhibition of cell proliferation and increase in cytochrome c release via perturbation in mitochondrial membrane potential (Δψm). Further, YK up-regulated the expression of tumor suppressor p53 and key cyclin-dependent kinase inhibitor p21, and inhibited VEGF secretion by cells. Interestingly, YK also exhibited a synergy with paclitaxel which is a well-known anti-cancer therapeutic drug.

Conclusions: The pharmacological properties of YK to impose growth arrest and trigger pro-apoptotic death in cells amply justify its usage in primary as well as adjunct therapy for ovarian cancer.

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Related in: MedlinePlus

Alterations in mitochondrial functions followingYKtreatment of SKOV6 cells. A, Confocal images of JC1 stained cells treated with YK for 24 h. The green fluorescence shows depolarized mitochondria (monomer), whereas red fluorescence shows hyperpolarized (aggregates) mitochondria. B, Cells treated with YK for 24 h showing the release of cytochrome c from mitochondria during apoptosis. Cells were co-transfected with cytochrome c-GFP fusion construct and dsRed mitotracker and visualized by confocal microscopy. Green represents cytochrome c, red mitochondria and orange indicates co-localization of cytochrome c and mitochondria. Results in both panels are represented as mean of three independent experiments ± SD Level of significance; *, p <0.05; **, p <0.001.
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Fig3: Alterations in mitochondrial functions followingYKtreatment of SKOV6 cells. A, Confocal images of JC1 stained cells treated with YK for 24 h. The green fluorescence shows depolarized mitochondria (monomer), whereas red fluorescence shows hyperpolarized (aggregates) mitochondria. B, Cells treated with YK for 24 h showing the release of cytochrome c from mitochondria during apoptosis. Cells were co-transfected with cytochrome c-GFP fusion construct and dsRed mitotracker and visualized by confocal microscopy. Green represents cytochrome c, red mitochondria and orange indicates co-localization of cytochrome c and mitochondria. Results in both panels are represented as mean of three independent experiments ± SD Level of significance; *, p <0.05; **, p <0.001.

Mentions: Since, mitochondria plays a key role in energy metabolism, and proving to be the novel target in killing cancerous cells [19], we sought to determine the effect of YK on mitochondrial function with the help of cationic JC1 dye. Exposure of cells to YK led to the disappearance of red fluorescence and increase in the green fluorescence in most cells combined with a significant reduction (p <0.001) in mitochondrial membrane potential. (Figure 3A) Further, YK along with Paclitaxel perturbed mitochondrial membrane potential to a significant level (p <0.05) as compared to only paclitaxel treatment (Figure 3B).Figure 3


Molecular insights into the anti-cancer properties of traditional Tibetan medicine Yukyung Karne.

Choedon T, Dolma D, Mathan G, Kumar V - BMC Complement Altern Med (2014)

Alterations in mitochondrial functions followingYKtreatment of SKOV6 cells. A, Confocal images of JC1 stained cells treated with YK for 24 h. The green fluorescence shows depolarized mitochondria (monomer), whereas red fluorescence shows hyperpolarized (aggregates) mitochondria. B, Cells treated with YK for 24 h showing the release of cytochrome c from mitochondria during apoptosis. Cells were co-transfected with cytochrome c-GFP fusion construct and dsRed mitotracker and visualized by confocal microscopy. Green represents cytochrome c, red mitochondria and orange indicates co-localization of cytochrome c and mitochondria. Results in both panels are represented as mean of three independent experiments ± SD Level of significance; *, p <0.05; **, p <0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4197273&req=5

Fig3: Alterations in mitochondrial functions followingYKtreatment of SKOV6 cells. A, Confocal images of JC1 stained cells treated with YK for 24 h. The green fluorescence shows depolarized mitochondria (monomer), whereas red fluorescence shows hyperpolarized (aggregates) mitochondria. B, Cells treated with YK for 24 h showing the release of cytochrome c from mitochondria during apoptosis. Cells were co-transfected with cytochrome c-GFP fusion construct and dsRed mitotracker and visualized by confocal microscopy. Green represents cytochrome c, red mitochondria and orange indicates co-localization of cytochrome c and mitochondria. Results in both panels are represented as mean of three independent experiments ± SD Level of significance; *, p <0.05; **, p <0.001.
Mentions: Since, mitochondria plays a key role in energy metabolism, and proving to be the novel target in killing cancerous cells [19], we sought to determine the effect of YK on mitochondrial function with the help of cationic JC1 dye. Exposure of cells to YK led to the disappearance of red fluorescence and increase in the green fluorescence in most cells combined with a significant reduction (p <0.001) in mitochondrial membrane potential. (Figure 3A) Further, YK along with Paclitaxel perturbed mitochondrial membrane potential to a significant level (p <0.05) as compared to only paclitaxel treatment (Figure 3B).Figure 3

Bottom Line: Confocal microscopy suggested inhibition of cell proliferation and increase in cytochrome c release via perturbation in mitochondrial membrane potential (Δψm).Further, YK up-regulated the expression of tumor suppressor p53 and key cyclin-dependent kinase inhibitor p21, and inhibited VEGF secretion by cells.Interestingly, YK also exhibited a synergy with paclitaxel which is a well-known anti-cancer therapeutic drug.

View Article: PubMed Central - PubMed

Affiliation: Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. vijay@icgeb.res.in.

ABSTRACT

Background: Yukyung karne (YK) is a traditional Tibetan formulation used for many centuries for the treatment of ovarian cancer. However, the pharmacological basis of its anticancer property is not well understood. In the present study, the anticancer property of YK was investigated in cell culture.

Methods: The growth inhibitory property of YK was evaluated in SKOV6, IHH, HepG2 and HEK293 cell lines using MTT assay. The pro-apoptotic activity of drug was analyzed by terminal deoxynuleotidyl transferase dUTP nick end labeling (TUNEL) and DNA fragmentation assays. Confocal microscopy was used to show the release of cytochrome c and its co-localization with mitochondria with the help of dsRed mitotracker in SKOV6 cells. The inhibition in cell proliferation was also visualized by confocal microscopy after BrDU incorporation. The activation of tumor suppressor p53 was evaluated by Western blotting while VEGF levels in culture supernatant were measured by a colorimetric method.

Results: YK specifically and efficiently induced apoptotic killing of the human ovarian cancer SKOV6 cells as indicated by increased DNA fragmentation and nick end DNA labeling. Confocal microscopy suggested inhibition of cell proliferation and increase in cytochrome c release via perturbation in mitochondrial membrane potential (Δψm). Further, YK up-regulated the expression of tumor suppressor p53 and key cyclin-dependent kinase inhibitor p21, and inhibited VEGF secretion by cells. Interestingly, YK also exhibited a synergy with paclitaxel which is a well-known anti-cancer therapeutic drug.

Conclusions: The pharmacological properties of YK to impose growth arrest and trigger pro-apoptotic death in cells amply justify its usage in primary as well as adjunct therapy for ovarian cancer.

Show MeSH
Related in: MedlinePlus