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Apoptosis of Hepatocellular Carcinoma Cells Induced by Nanoencapsulated Polysaccharides Extracted from Antrodia Camphorata.

Chang JS, Kuo HP, Chang KL, Kong ZL - PLoS ONE (2015)

Bottom Line: In this study, ACE polysaccharides were nano-encapsulated in chitosan-silica and silica (expressed as ACE/CS and ACE/S, respectively) to evaluate the apoptosis effect on a hepatoma cell line (Hep G2).The results showed that ACE polysaccharides, ACE/CS and ACE/S all could damage the Hep G2 cell membrane and cause cell death, especially in the ACE/CS group.In apoptosis assays, DNA fragmentation and sub-G1 phase populations were increased, and the mitochondrial membrane potential decreased significantly after treatments.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan.

ABSTRACT
Antrodia camphorata is a well-known medicinal mushroom in Taiwan and has been studied for decades, especially with focus on anti-cancer activity. Polysaccharides are the major bioactive compounds reported with anti-cancer activity, but the debates on how they target cells still remain. Research addressing the encapsulation of polysaccharides from A. camphorata extract (ACE) to enhance anti-cancer activity is rare. In this study, ACE polysaccharides were nano-encapsulated in chitosan-silica and silica (expressed as ACE/CS and ACE/S, respectively) to evaluate the apoptosis effect on a hepatoma cell line (Hep G2). The results showed that ACE polysaccharides, ACE/CS and ACE/S all could damage the Hep G2 cell membrane and cause cell death, especially in the ACE/CS group. In apoptosis assays, DNA fragmentation and sub-G1 phase populations were increased, and the mitochondrial membrane potential decreased significantly after treatments. ACE/CS and ACE/S could also increase reactive oxygen species (ROS) generation, induce Fas/APO-1 (apoptosis antigen 1) expression and elevate the proteolytic activities of caspase-3, caspase-8 and caspase-9 in Hep G2 cells. Unsurprisingly, ACE/CS induced a similar apoptosis mechanism at a lower dosage (ACE polysaccharides = 13.2 μg/mL) than those of ACE/S (ACE polysaccharides = 21.2 μg/mL) and ACE polysaccharides (25 μg/mL). Therefore, the encapsulation of ACE polysaccharides by chitosan-silica nanoparticles may provide a viable approach for enhancing anti-tumor efficacy in liver cancer cells.

No MeSH data available.


Related in: MedlinePlus

The DNA electrophoresis of Hep G2 cells after treatment with ACE polysaccharides, ACE/CS and ACE/S for 48 h.Hep G2 cells were seeded at the density of 2.0 × 105 cells/mL in 10-cm dishes. After different treatments, cells were harvested, lysed and centrifuged at 14,000× g for supernatant collection. The fragmented DNAs were extracted, analyzed with 2% agarose gel electrophoresis containing 0.1 μg/mL ethidium bromide and imaged. Experiments were repeated 3 times independently to ensure reproducibility. M: Maker, A: 25 μg/mL ACE polysaccharides, B: ACE/CS (ACE polysaccharides = 13.2 μg/mL), C: ACE/S (ACE polysaccharides = 21.2 μg/mL), D: 100 μg/mL Trail for positive control and Con: without any treatment for control. ACE: A. camphorata extract; ACE/CS: ACE polysaccharides encapsulated by chitosan-silica nanoparticles; ACE/S: ACE polysaccharides encapsulated by silica nanoparticles.
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pone.0136782.g004: The DNA electrophoresis of Hep G2 cells after treatment with ACE polysaccharides, ACE/CS and ACE/S for 48 h.Hep G2 cells were seeded at the density of 2.0 × 105 cells/mL in 10-cm dishes. After different treatments, cells were harvested, lysed and centrifuged at 14,000× g for supernatant collection. The fragmented DNAs were extracted, analyzed with 2% agarose gel electrophoresis containing 0.1 μg/mL ethidium bromide and imaged. Experiments were repeated 3 times independently to ensure reproducibility. M: Maker, A: 25 μg/mL ACE polysaccharides, B: ACE/CS (ACE polysaccharides = 13.2 μg/mL), C: ACE/S (ACE polysaccharides = 21.2 μg/mL), D: 100 μg/mL Trail for positive control and Con: without any treatment for control. ACE: A. camphorata extract; ACE/CS: ACE polysaccharides encapsulated by chitosan-silica nanoparticles; ACE/S: ACE polysaccharides encapsulated by silica nanoparticles.

Mentions: The characteristic DNA laddering of apoptosis was carried out in Hep G2 cells treated with ACE polysaccharides, ACE/CS and ACE/S at the concentrations of 25 μg/mL, 13.2 μg/mL and 21.2 μg/mL, respectively, by using agarose gel electrophoresis. The results showed a ladder-like pattern of multiple DNA fragments consisting of approximately 180–200 base pairs (Fig 4). In cell cycle analysis, flow cytometry for quantifying the DNA fragmentation extent was used and the apoptotic cells were identified as the hypodiploidy peak (sub-G1) on the PI histogram. The percentage of apoptotic cells was 1.42% in the control group and reached 1.46% after treatment with ACE polysaccharides (25 μg/mL) in 24 h (Table 1 and S1 Fig). When cells were incubated with ACE/S (containing 21.2 μg/mL ACE polysaccharides) for 24 h, the sub-G1 phase population slightly increased to 2.76%, whereas it increased to 2.88% for the cells incubated in ACE/CS (containing 13.2 μg/mL ACE polysaccharides; Table 1 and S1 Fig). After 48 h incubation, the sub-G1 phase population approached 5.18% in the control group and reached 20.98% or 20.56% in ACE polysaccharides or ACE/CS group, respectively, but only increased to 10.86% for cells incubated with ACE/S (Table 1 and S1 Fig). In contrast, after Hep G2 was incubated with silica and chitosan-silica nanoparticles without ACE polysaccharides even at the highest concentration (667 μg/mL) for 48 h, the sub-G1 phase population was just slightly increased to 6.54% and 9.16% (Table 1).


Apoptosis of Hepatocellular Carcinoma Cells Induced by Nanoencapsulated Polysaccharides Extracted from Antrodia Camphorata.

Chang JS, Kuo HP, Chang KL, Kong ZL - PLoS ONE (2015)

The DNA electrophoresis of Hep G2 cells after treatment with ACE polysaccharides, ACE/CS and ACE/S for 48 h.Hep G2 cells were seeded at the density of 2.0 × 105 cells/mL in 10-cm dishes. After different treatments, cells were harvested, lysed and centrifuged at 14,000× g for supernatant collection. The fragmented DNAs were extracted, analyzed with 2% agarose gel electrophoresis containing 0.1 μg/mL ethidium bromide and imaged. Experiments were repeated 3 times independently to ensure reproducibility. M: Maker, A: 25 μg/mL ACE polysaccharides, B: ACE/CS (ACE polysaccharides = 13.2 μg/mL), C: ACE/S (ACE polysaccharides = 21.2 μg/mL), D: 100 μg/mL Trail for positive control and Con: without any treatment for control. ACE: A. camphorata extract; ACE/CS: ACE polysaccharides encapsulated by chitosan-silica nanoparticles; ACE/S: ACE polysaccharides encapsulated by silica nanoparticles.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556685&req=5

pone.0136782.g004: The DNA electrophoresis of Hep G2 cells after treatment with ACE polysaccharides, ACE/CS and ACE/S for 48 h.Hep G2 cells were seeded at the density of 2.0 × 105 cells/mL in 10-cm dishes. After different treatments, cells were harvested, lysed and centrifuged at 14,000× g for supernatant collection. The fragmented DNAs were extracted, analyzed with 2% agarose gel electrophoresis containing 0.1 μg/mL ethidium bromide and imaged. Experiments were repeated 3 times independently to ensure reproducibility. M: Maker, A: 25 μg/mL ACE polysaccharides, B: ACE/CS (ACE polysaccharides = 13.2 μg/mL), C: ACE/S (ACE polysaccharides = 21.2 μg/mL), D: 100 μg/mL Trail for positive control and Con: without any treatment for control. ACE: A. camphorata extract; ACE/CS: ACE polysaccharides encapsulated by chitosan-silica nanoparticles; ACE/S: ACE polysaccharides encapsulated by silica nanoparticles.
Mentions: The characteristic DNA laddering of apoptosis was carried out in Hep G2 cells treated with ACE polysaccharides, ACE/CS and ACE/S at the concentrations of 25 μg/mL, 13.2 μg/mL and 21.2 μg/mL, respectively, by using agarose gel electrophoresis. The results showed a ladder-like pattern of multiple DNA fragments consisting of approximately 180–200 base pairs (Fig 4). In cell cycle analysis, flow cytometry for quantifying the DNA fragmentation extent was used and the apoptotic cells were identified as the hypodiploidy peak (sub-G1) on the PI histogram. The percentage of apoptotic cells was 1.42% in the control group and reached 1.46% after treatment with ACE polysaccharides (25 μg/mL) in 24 h (Table 1 and S1 Fig). When cells were incubated with ACE/S (containing 21.2 μg/mL ACE polysaccharides) for 24 h, the sub-G1 phase population slightly increased to 2.76%, whereas it increased to 2.88% for the cells incubated in ACE/CS (containing 13.2 μg/mL ACE polysaccharides; Table 1 and S1 Fig). After 48 h incubation, the sub-G1 phase population approached 5.18% in the control group and reached 20.98% or 20.56% in ACE polysaccharides or ACE/CS group, respectively, but only increased to 10.86% for cells incubated with ACE/S (Table 1 and S1 Fig). In contrast, after Hep G2 was incubated with silica and chitosan-silica nanoparticles without ACE polysaccharides even at the highest concentration (667 μg/mL) for 48 h, the sub-G1 phase population was just slightly increased to 6.54% and 9.16% (Table 1).

Bottom Line: In this study, ACE polysaccharides were nano-encapsulated in chitosan-silica and silica (expressed as ACE/CS and ACE/S, respectively) to evaluate the apoptosis effect on a hepatoma cell line (Hep G2).The results showed that ACE polysaccharides, ACE/CS and ACE/S all could damage the Hep G2 cell membrane and cause cell death, especially in the ACE/CS group.In apoptosis assays, DNA fragmentation and sub-G1 phase populations were increased, and the mitochondrial membrane potential decreased significantly after treatments.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan.

ABSTRACT
Antrodia camphorata is a well-known medicinal mushroom in Taiwan and has been studied for decades, especially with focus on anti-cancer activity. Polysaccharides are the major bioactive compounds reported with anti-cancer activity, but the debates on how they target cells still remain. Research addressing the encapsulation of polysaccharides from A. camphorata extract (ACE) to enhance anti-cancer activity is rare. In this study, ACE polysaccharides were nano-encapsulated in chitosan-silica and silica (expressed as ACE/CS and ACE/S, respectively) to evaluate the apoptosis effect on a hepatoma cell line (Hep G2). The results showed that ACE polysaccharides, ACE/CS and ACE/S all could damage the Hep G2 cell membrane and cause cell death, especially in the ACE/CS group. In apoptosis assays, DNA fragmentation and sub-G1 phase populations were increased, and the mitochondrial membrane potential decreased significantly after treatments. ACE/CS and ACE/S could also increase reactive oxygen species (ROS) generation, induce Fas/APO-1 (apoptosis antigen 1) expression and elevate the proteolytic activities of caspase-3, caspase-8 and caspase-9 in Hep G2 cells. Unsurprisingly, ACE/CS induced a similar apoptosis mechanism at a lower dosage (ACE polysaccharides = 13.2 μg/mL) than those of ACE/S (ACE polysaccharides = 21.2 μg/mL) and ACE polysaccharides (25 μg/mL). Therefore, the encapsulation of ACE polysaccharides by chitosan-silica nanoparticles may provide a viable approach for enhancing anti-tumor efficacy in liver cancer cells.

No MeSH data available.


Related in: MedlinePlus