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

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.

Figure 1

Growth inhibitory effect of YK on different cell lines. A. HEK293T, A549, HepG2, Hela, IHH and SKOV6 cells were treated with different doses of YK (1, 10, 100 μg/ml) and analyzed for cell viability by MTT assay. B. SKOV6 cells was treated with YK and/ or paclitaxel (10 nM) and the cell viability was measured as above. Results are represented as mean of three independent experiments ± S.E.M. Level of significance; *, p <0.005.

Figure 2

Induction of apoptosis by YK in SKOV6 ovarian cells. A, Cells were treated with indicated doses of YK and/or paclitaxel [Lanes: 1-Control, 2-Paclitaxel 10nM, 3-Pac + YK (10nM + 100 μg/ml), 4–7 YK (1, 10, 100, and 200 μg/ml respectively) and 8-50 bp ladder] for 24 h. Genomic DNA was isolated and resolved by agarose gel electrophoresis. B, Cells were treated with different doses of YK (1, 10, 100 μg/ml) was visualized by TUNEL assay. The TUNEL positive cells (stained by FITC) were counted from random fields and are presented in the graph. Nuclei were visualized by DAPI staining (blue). Results are represented as mean of three independent experiments ± SD. Level of significance; *, p <0.001. C, Induction of cell cycle regulators. SKOV6 cells were treated for 24 h with YK (100 μg/ml), Paclitaxel (Pac) (10 μM) or both and the expression of p21, p53, Mdm2, cyclin B1, and PCNA was verified by western blot. GAPDH was used as internal control.

Figure 3

Alterations in mitochondrial functions following YK treatment 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.

Figure 4

Inhibition of cell proliferation in the presence of YK. SKOV 6 cells were treated with paclitaxel (10nM) and/or YK 100 μg/ml for 24 h and analyzed by confocal microscopy for BrdU incorporation for DNA synthesis. Results are represented as mean of three independent experiments ± SD. Level of significance; *, p <0.02, **, p <0.002.