Biol. Pharm. Bull. 38, 102–108 (2015)
102
Vol. 38, No. 1
Regular Article
Stereoisomer-Specific Anticancer Activities of Ginsenoside Rg3 and Rh2 in HepG2 Cells: Disparity in Cytotoxicity and Autophagy-Inducing Effects Due to 20(S)-Epimers Jong Hye Cheong,a Hyeryung Kim,a Min Jee Hong,a Min Hye Yang,a Jung Wha Kim,a Hunseung Yoo,a Heejung Yang,a Jeong Hill Park,a Sang Hyun Sung,a Hong Pyo Kim,*,b and Jinwoong Kim*,a a
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University; Seoul 151–742, Korea: and b School of Pharmacy, Ajou University; Suwon 442–721, Korea. Received August 21, 2014; accepted October 4, 2014 Autophagy has been an emerging field in the treatment of hepatic carcinoma since anticancer therapies were shown to ignite autophagy in vitro and in vivo. Here we report that ginsenoside Rg3 and Rh2, major components of red ginseng, induce apoptotic cell death in a stereoisomer-specific fashion. The 20(S)-forms of Rg3 and Rh2, but not their respective 20(R)-forms, promoted cell death in a dose-dependent manner accompanied by downregulation of Bcl2 and upregulation of Fas, resulting in apoptosis of HepG2 cells with poly ADP ribose polymerase cleavage. The LD50 value [45 µM for Rg3(S), less than 10 µM for Rh2(S)] and gross morphological electron microscopic observation revealed more severe cellular damage in cells treated with Rh2(S) than in those treated with Rg3(S). Both Rg3(S) and Rh2(S) also induced autophagy when undergoing induced apoptosis. Inhibition of autophagy with lysosomotrophic agents significantly potentiated the cellular damage, implying a favorable switch of the cell fate to tumor cell death. Blocking intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) restored the cell death induced by both Rg3(S) and Rh2(S). Our results suggest that the 20(S)-forms of Rg3 and Rh2 in red ginseng possess more potent antitumor activity with autophagy than their 20(R)-forms via calcium-dependent apoptosis. Key words
ginsenoside Rg3; ginsenoside Rh2; red ginseng; stereoisomer; autophagy; HepG2
Autophagy is an evolutionarily conserved intracellular degradation process that delivers cytoplasmic constituents to lysosomes via double-membrane vesicles termed autophagosomes. It contributes to the turnover of long-lived proteins and organelles to maintain cell homeostasis, and those upregulated under cellular stress conditions such as nutrient depletion, hypoxia and anticancer treatments.1) In cancer cells, it is known that autophagy can lead to autophagic cell death, or can promote cancer cell survival via protective mechanism. Indeed, this double-edged role of autophagy often determines the fate of the cell as it is the balance between cell death and survival. However, the role of autophagy in cancer chemotherapy is still controversial.2,3) Red ginseng is the steam-processed form of Panax ginseng roots, and has been widely used in Asian countries as a tonic medicine. Its major bioactive components, ginsenosides, are saponins with dammarane skeletons that have diverse biological activity including antiinflammatory, anti-allergic, anticancer, anti-viral, anti-diabetic, anti-obesity, and antihypertension effects4–6) in spite of their low bioavailabilities, which are mainly due to their metabolism in the gastrointestinal tract. Ginsenoside has a hydrophobic triterpenoidal backbone to which hydrophilic sugar moieties are attached at the carbon-3, −6, and −20 positions. It is well known that the structural differences of ginsenosides, with respect to the numbers and positions of sugar molecules, hydroxyl groups in dammarane skeleton, and stereoisomerism at C-20, influence the biological activity of each ginsenoside.7–9) Among them, protopanaxadiol type ginsenoside Rg3 and Rh2, are reported to have cell growth inhibitory effects in various cancer cells.10,11) They
possess one or two sugar units at C-3 and exist as stereoisomers depending on the position of the hydroxyl group at the C-20 position like other ginsenosides (Fig. 1A). Although stereospecificity is important in the recognition of substrate molecules by tertiary-structural binding sites in drug action, only a few reports focus on the differences in biological activity of the epimers of ginsenoside.12–14) In this study, we compared cytotoxic and autophagy-inducing activities between 20(S)- and 20(R)-epimers of ginsenoside Rg3 and Rh2 in hepatocellular carcinoma HepG2 cells.
MATERIALS AND METHODS Cell Culture Human hepatocellular carcinoma cell line HepG2 was purchased from the Korean Cell Line Bank (KCLB, Korea). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose medium (Sigma, St. Louis, MO, U.S.A.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone, Logan, UT, U.S.A.) along with penicillin (100 units/mL) and streptomycin (100 µg/mL) (Sigma) at 37°C in a humidified atmosphere of 5% CO2. Male Sprague-Dawley (SD) rats were obtained from Koatech Co., Ltd. (Yongin, Korea) with body weight of 200–250 g. Primary rat hepatocytes were isolated from SD male rats using the collagenase perfusion technique. The freshly isolated hepatocytes were suspended in DMEM/high glucose medium containing 10% FBS, 1 µM dexamethasone (Sigma), 0.1 µM insulin (Sigma), 100 U/mL penicillin and 100 µg/mL streptomycin. Cells were inoculated on rat tail collagen-coated 35 mm×10 mm style culture dishes (Corning, NY, U.S.A.) at a
* To whom correspondence should be addressed. e-mail: [email protected]; [email protected] © 2015 The Pharmaceutical Society of Japan
Biol. Pharm. Bull. Vol. 38, No. 1 (2015)103
Fig. 1. (A) Chemical Structures of Ginsenoside Rg3 and Rh2; (B) 20(S)-Ginsenoside Rg3 and Rh2 Induce Cytotoxicity in HepG2 Cancer Cell; (C) 20(S)-Ginsenoside Rg3 and Rh2 Show No Cytotoxicity in Rat Primary Hepatocyte Cells were treated with the indicated concentration for 24 h in serum-free DMEM media. Cell viability was analyzed by MTT assay. Values represent the mean±S.D. of three independent experiments.
density of 2×105 cells/mL and maintained in a humidified incubator containing 5% CO2 gas at 37°C until cell attachment. Measurement of Cell Viability The cell growth inhibitory activities on HepG2 cells and rat primary hepatocyte were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) colorimetric assay. Cells were treated with vehicle control dimethyl sulfoxide (DMSO) or compounds for 24 h with or without autophagy inhibitor, chloroquine (20 µM chloroquine diphosphate salt, ≥98%, Sigma-Aldrich) then added in solution with MTT (0.5 mg/mL; Sigma-Aldrich) to each well. After incubation for 4 h at 37°C the supernatant was aspirated, and the MTT-formazan crystals formed by the metabolically viable cells were dissolved in DMSO. The absorbance was measured by enzyme-linked immunosorbent assay (ELISA) at a wavelength of 540 nm. To investigate the anti-proliferative effect, the cells were incubated for 24 h in the various concentrations of Rg3 and Rh2 treated with or without 20 µM chloroquine (chloroquine diphosphate salt, ≥98%, Sigma-Aldrich) and 10 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), ≥95%, Sigma-Aldrich). Transmission Electron Microscopy (TEM) Cells were treated with 50 µM Rg3 and 20 µM Rh2 for 24 h and collected by trypsinization, fixed with Karnovsky’s fixation reagent primarily, and postfixed in 2% cacodylate-buffered osmium tetroxide. Cells were then stained with 0.5% uranyl acetate, embedded, sectioned, and analyzed using a JEM-1010 transmission electron microscope (JEOL, Tokyo, Japan). Fluorescence Microscopy In order to detect acidic autophagic vacuoles, we used the autofluorescent marker monodansylcadaverine (MDC). After incubation with 25 µM Rg3 and Rh2 for 24 h, cells were labeled with 50 µM MDC at 37°C for 30 min, washed 3 times with PBS and mounted. Images were obtained at 400× magnification at the excitation wavelength 330–385 nm with an emission filter at 420 nm. Western Blot Cells were lysed with sodium dodecyl sulfate (SDS) lysis buffer containing a protease inhibitor
cocktail. The lysates were centrifuged and supernatants collected. Proteins were separated in 8–15% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane. Membrane was blocked in TBS containing 0.1% Tween 20 (TBST) and 5% nonfat dry milk for 1 h at room temperature and then incubated overnight with primary antibodies in TBST containing 1% nonfat dry milk at 4°C. Membranes were then washed with TBST and incubated with goat anti-rabbit (donkey anti-goat for UCP-2 detection) peroxidase conjugated immunoglobulin G (IgG) secondary antibody for 2 h, and immune complexes were detected by enhanced chemiluminescence. Primary antibodies used in Western blot were goat anti-UCP-2 (Santa Cruz) and rabbit anti-LC3B (Sigma-Aldrich), anti-cleaved PARP (Cell Signaling), anti-Bcl-2, anti-Fas, anti-HO-1, anti-TFEB, anti-ATPIF-1 (Santa Cruz), anti-OPA-1 (Novus Biologicals) and β-actin (Abfrontier). The intensities of bands were quantified by ImageQuant TL (GE Healthcare) software. Cells were incubated in 20 µM of Rh2 and 50 µM of Rg3 for 8–16 h, then harvested. Ammonium chloride (NH4Cl 10 m M), a lysosomotropic agent, was added to cultural medium to block the proteolysis of LC3 II. Flow Cytometry To analyze the cell cycle distribution and the mode of dell death, cells were plated in 6-well plates and treated with 50 µM Rg3(S) and 20 µM Rh2(S) with and without 20 µM chloroquine for 24 h. For cell cycle analysis, cells were harvested, washed with phosphate buffered saline (PBS), and fixed in 80% EtOH at 4°C overnight. They were then washed with PBS and resuspended in 500 µL of propidium iodide (PI) staining solution containing 40 µg/mL PI and 20 µg/mL RNase A in PBS, incubated at room temperature for 30 min in the dark, and then analyzed with FACSCaliburTM flow cytometer (BD Science, U.S.A.). For quantitative determination of apoptosis and necrosis, harvested cells were washed with PBS, diluted in annexin V binding buffer containing annexin V and PI, incubated for 15 min in the dark, and analyzed with flow cytometer. Data from 10000 cells per
104
Fig. 2.
Biol. Pharm. Bull.
Vol. 38, No. 1 (2015)
20(S)-Ginsenoside Rg3 and Rh2 Induce Apoptotic Death in HepG2 Cells
Cells were treated with the indicated concentration for 24h in serum-free DMEM media. (A) Cells were treated in 50 µM Rg3(S) and 20 µM Rh2(S), then harvested and double-stained with annexin V and PI followed by analyzing with flow cytometer (FL1: annexin V, FL2: PI). (B) Microscopic images after 16 h treatment of 50 µM Rg3 and 20 µM Rh2 (×400).
Fig. 3.
20(S)-Ginsenoside Rg3 and Rh2 Induce Mitochondrial Damage with Apoptotic Death in HepG2 Cells
Cells were treated with 50 µM Rg3 and 20 µM Rh2 in serum-free DMEM media then analyzed. (A) (B) TEM images were obtained after ginsenoside treatment for 16 h. Mitochondrial abnormality by 20(S)-ginsenoside Rg3 and Rh2 treatment are indicated with black arrows. (C) Expression levels of bcl-2, Fas, HO-1 and cleaved PARP were estimated by immunoblotting after treatment of ginsenosides. (D) Numbers of mitochondria in HepG2 cell assorted by size. (E) Expression levels of OPA-1, UCP-2 and ATPIF-1 were evaluated by Western blotting after 16 h treatment of ginsenosides. β-Actin was used as the internal control.
Biol. Pharm. Bull. Vol. 38, No. 1 (2015)105
Fig. 4.
20(S)-Ginsenoside Rg3 and Rh2 Induce Autophagy in HepG2 Cells
Cells were treated with 25 µM Rg3 and Rh2 for 24 h (8 or 16 h for Western blot) in serum-free DMEM media then analyzed. (A) Cells were stained with MDC and observed under fluorescence microscope. The numbers of MDC-labeled autophagic vacuoles per 100 cells compared with DMSO control were presented in the histogram. Values represent the mean±S.D. of three independent experiments (* p
102
Vol. 38, No. 1
Regular Article
Stereoisomer-Specific Anticancer Activities of Ginsenoside Rg3 and Rh2 in HepG2 Cells: Disparity in Cytotoxicity and Autophagy-Inducing Effects Due to 20(S)-Epimers Jong Hye Cheong,a Hyeryung Kim,a Min Jee Hong,a Min Hye Yang,a Jung Wha Kim,a Hunseung Yoo,a Heejung Yang,a Jeong Hill Park,a Sang Hyun Sung,a Hong Pyo Kim,*,b and Jinwoong Kim*,a a
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University; Seoul 151–742, Korea: and b School of Pharmacy, Ajou University; Suwon 442–721, Korea. Received August 21, 2014; accepted October 4, 2014 Autophagy has been an emerging field in the treatment of hepatic carcinoma since anticancer therapies were shown to ignite autophagy in vitro and in vivo. Here we report that ginsenoside Rg3 and Rh2, major components of red ginseng, induce apoptotic cell death in a stereoisomer-specific fashion. The 20(S)-forms of Rg3 and Rh2, but not their respective 20(R)-forms, promoted cell death in a dose-dependent manner accompanied by downregulation of Bcl2 and upregulation of Fas, resulting in apoptosis of HepG2 cells with poly ADP ribose polymerase cleavage. The LD50 value [45 µM for Rg3(S), less than 10 µM for Rh2(S)] and gross morphological electron microscopic observation revealed more severe cellular damage in cells treated with Rh2(S) than in those treated with Rg3(S). Both Rg3(S) and Rh2(S) also induced autophagy when undergoing induced apoptosis. Inhibition of autophagy with lysosomotrophic agents significantly potentiated the cellular damage, implying a favorable switch of the cell fate to tumor cell death. Blocking intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) restored the cell death induced by both Rg3(S) and Rh2(S). Our results suggest that the 20(S)-forms of Rg3 and Rh2 in red ginseng possess more potent antitumor activity with autophagy than their 20(R)-forms via calcium-dependent apoptosis. Key words
ginsenoside Rg3; ginsenoside Rh2; red ginseng; stereoisomer; autophagy; HepG2
Autophagy is an evolutionarily conserved intracellular degradation process that delivers cytoplasmic constituents to lysosomes via double-membrane vesicles termed autophagosomes. It contributes to the turnover of long-lived proteins and organelles to maintain cell homeostasis, and those upregulated under cellular stress conditions such as nutrient depletion, hypoxia and anticancer treatments.1) In cancer cells, it is known that autophagy can lead to autophagic cell death, or can promote cancer cell survival via protective mechanism. Indeed, this double-edged role of autophagy often determines the fate of the cell as it is the balance between cell death and survival. However, the role of autophagy in cancer chemotherapy is still controversial.2,3) Red ginseng is the steam-processed form of Panax ginseng roots, and has been widely used in Asian countries as a tonic medicine. Its major bioactive components, ginsenosides, are saponins with dammarane skeletons that have diverse biological activity including antiinflammatory, anti-allergic, anticancer, anti-viral, anti-diabetic, anti-obesity, and antihypertension effects4–6) in spite of their low bioavailabilities, which are mainly due to their metabolism in the gastrointestinal tract. Ginsenoside has a hydrophobic triterpenoidal backbone to which hydrophilic sugar moieties are attached at the carbon-3, −6, and −20 positions. It is well known that the structural differences of ginsenosides, with respect to the numbers and positions of sugar molecules, hydroxyl groups in dammarane skeleton, and stereoisomerism at C-20, influence the biological activity of each ginsenoside.7–9) Among them, protopanaxadiol type ginsenoside Rg3 and Rh2, are reported to have cell growth inhibitory effects in various cancer cells.10,11) They
possess one or two sugar units at C-3 and exist as stereoisomers depending on the position of the hydroxyl group at the C-20 position like other ginsenosides (Fig. 1A). Although stereospecificity is important in the recognition of substrate molecules by tertiary-structural binding sites in drug action, only a few reports focus on the differences in biological activity of the epimers of ginsenoside.12–14) In this study, we compared cytotoxic and autophagy-inducing activities between 20(S)- and 20(R)-epimers of ginsenoside Rg3 and Rh2 in hepatocellular carcinoma HepG2 cells.
MATERIALS AND METHODS Cell Culture Human hepatocellular carcinoma cell line HepG2 was purchased from the Korean Cell Line Bank (KCLB, Korea). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose medium (Sigma, St. Louis, MO, U.S.A.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone, Logan, UT, U.S.A.) along with penicillin (100 units/mL) and streptomycin (100 µg/mL) (Sigma) at 37°C in a humidified atmosphere of 5% CO2. Male Sprague-Dawley (SD) rats were obtained from Koatech Co., Ltd. (Yongin, Korea) with body weight of 200–250 g. Primary rat hepatocytes were isolated from SD male rats using the collagenase perfusion technique. The freshly isolated hepatocytes were suspended in DMEM/high glucose medium containing 10% FBS, 1 µM dexamethasone (Sigma), 0.1 µM insulin (Sigma), 100 U/mL penicillin and 100 µg/mL streptomycin. Cells were inoculated on rat tail collagen-coated 35 mm×10 mm style culture dishes (Corning, NY, U.S.A.) at a
* To whom correspondence should be addressed. e-mail: [email protected]; [email protected] © 2015 The Pharmaceutical Society of Japan
Biol. Pharm. Bull. Vol. 38, No. 1 (2015)103
Fig. 1. (A) Chemical Structures of Ginsenoside Rg3 and Rh2; (B) 20(S)-Ginsenoside Rg3 and Rh2 Induce Cytotoxicity in HepG2 Cancer Cell; (C) 20(S)-Ginsenoside Rg3 and Rh2 Show No Cytotoxicity in Rat Primary Hepatocyte Cells were treated with the indicated concentration for 24 h in serum-free DMEM media. Cell viability was analyzed by MTT assay. Values represent the mean±S.D. of three independent experiments.
density of 2×105 cells/mL and maintained in a humidified incubator containing 5% CO2 gas at 37°C until cell attachment. Measurement of Cell Viability The cell growth inhibitory activities on HepG2 cells and rat primary hepatocyte were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) colorimetric assay. Cells were treated with vehicle control dimethyl sulfoxide (DMSO) or compounds for 24 h with or without autophagy inhibitor, chloroquine (20 µM chloroquine diphosphate salt, ≥98%, Sigma-Aldrich) then added in solution with MTT (0.5 mg/mL; Sigma-Aldrich) to each well. After incubation for 4 h at 37°C the supernatant was aspirated, and the MTT-formazan crystals formed by the metabolically viable cells were dissolved in DMSO. The absorbance was measured by enzyme-linked immunosorbent assay (ELISA) at a wavelength of 540 nm. To investigate the anti-proliferative effect, the cells were incubated for 24 h in the various concentrations of Rg3 and Rh2 treated with or without 20 µM chloroquine (chloroquine diphosphate salt, ≥98%, Sigma-Aldrich) and 10 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), ≥95%, Sigma-Aldrich). Transmission Electron Microscopy (TEM) Cells were treated with 50 µM Rg3 and 20 µM Rh2 for 24 h and collected by trypsinization, fixed with Karnovsky’s fixation reagent primarily, and postfixed in 2% cacodylate-buffered osmium tetroxide. Cells were then stained with 0.5% uranyl acetate, embedded, sectioned, and analyzed using a JEM-1010 transmission electron microscope (JEOL, Tokyo, Japan). Fluorescence Microscopy In order to detect acidic autophagic vacuoles, we used the autofluorescent marker monodansylcadaverine (MDC). After incubation with 25 µM Rg3 and Rh2 for 24 h, cells were labeled with 50 µM MDC at 37°C for 30 min, washed 3 times with PBS and mounted. Images were obtained at 400× magnification at the excitation wavelength 330–385 nm with an emission filter at 420 nm. Western Blot Cells were lysed with sodium dodecyl sulfate (SDS) lysis buffer containing a protease inhibitor
cocktail. The lysates were centrifuged and supernatants collected. Proteins were separated in 8–15% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane. Membrane was blocked in TBS containing 0.1% Tween 20 (TBST) and 5% nonfat dry milk for 1 h at room temperature and then incubated overnight with primary antibodies in TBST containing 1% nonfat dry milk at 4°C. Membranes were then washed with TBST and incubated with goat anti-rabbit (donkey anti-goat for UCP-2 detection) peroxidase conjugated immunoglobulin G (IgG) secondary antibody for 2 h, and immune complexes were detected by enhanced chemiluminescence. Primary antibodies used in Western blot were goat anti-UCP-2 (Santa Cruz) and rabbit anti-LC3B (Sigma-Aldrich), anti-cleaved PARP (Cell Signaling), anti-Bcl-2, anti-Fas, anti-HO-1, anti-TFEB, anti-ATPIF-1 (Santa Cruz), anti-OPA-1 (Novus Biologicals) and β-actin (Abfrontier). The intensities of bands were quantified by ImageQuant TL (GE Healthcare) software. Cells were incubated in 20 µM of Rh2 and 50 µM of Rg3 for 8–16 h, then harvested. Ammonium chloride (NH4Cl 10 m M), a lysosomotropic agent, was added to cultural medium to block the proteolysis of LC3 II. Flow Cytometry To analyze the cell cycle distribution and the mode of dell death, cells were plated in 6-well plates and treated with 50 µM Rg3(S) and 20 µM Rh2(S) with and without 20 µM chloroquine for 24 h. For cell cycle analysis, cells were harvested, washed with phosphate buffered saline (PBS), and fixed in 80% EtOH at 4°C overnight. They were then washed with PBS and resuspended in 500 µL of propidium iodide (PI) staining solution containing 40 µg/mL PI and 20 µg/mL RNase A in PBS, incubated at room temperature for 30 min in the dark, and then analyzed with FACSCaliburTM flow cytometer (BD Science, U.S.A.). For quantitative determination of apoptosis and necrosis, harvested cells were washed with PBS, diluted in annexin V binding buffer containing annexin V and PI, incubated for 15 min in the dark, and analyzed with flow cytometer. Data from 10000 cells per
104
Fig. 2.
Biol. Pharm. Bull.
Vol. 38, No. 1 (2015)
20(S)-Ginsenoside Rg3 and Rh2 Induce Apoptotic Death in HepG2 Cells
Cells were treated with the indicated concentration for 24h in serum-free DMEM media. (A) Cells were treated in 50 µM Rg3(S) and 20 µM Rh2(S), then harvested and double-stained with annexin V and PI followed by analyzing with flow cytometer (FL1: annexin V, FL2: PI). (B) Microscopic images after 16 h treatment of 50 µM Rg3 and 20 µM Rh2 (×400).
Fig. 3.
20(S)-Ginsenoside Rg3 and Rh2 Induce Mitochondrial Damage with Apoptotic Death in HepG2 Cells
Cells were treated with 50 µM Rg3 and 20 µM Rh2 in serum-free DMEM media then analyzed. (A) (B) TEM images were obtained after ginsenoside treatment for 16 h. Mitochondrial abnormality by 20(S)-ginsenoside Rg3 and Rh2 treatment are indicated with black arrows. (C) Expression levels of bcl-2, Fas, HO-1 and cleaved PARP were estimated by immunoblotting after treatment of ginsenosides. (D) Numbers of mitochondria in HepG2 cell assorted by size. (E) Expression levels of OPA-1, UCP-2 and ATPIF-1 were evaluated by Western blotting after 16 h treatment of ginsenosides. β-Actin was used as the internal control.
Biol. Pharm. Bull. Vol. 38, No. 1 (2015)105
Fig. 4.
20(S)-Ginsenoside Rg3 and Rh2 Induce Autophagy in HepG2 Cells
Cells were treated with 25 µM Rg3 and Rh2 for 24 h (8 or 16 h for Western blot) in serum-free DMEM media then analyzed. (A) Cells were stained with MDC and observed under fluorescence microscope. The numbers of MDC-labeled autophagic vacuoles per 100 cells compared with DMSO control were presented in the histogram. Values represent the mean±S.D. of three independent experiments (* p
