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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 cytotoxicity of ACE polysaccharides, ACE/CS and ACE/S on Hep G2 cells.Cells were incubated with samples for (a) 24 h or (b) 48 h and viability was assessed using an MTT assay. Nanoparticles without AEC polysaccharides were presented as (c) for 48 h incubation. Experiments were repeated 3 times independently to ensure reproducibility and the standard deviation of the mean are represented as error bars (n = 3). Values with different letters or asterisks were significantly different (p < 0.05) at corresponding concentrations between different treatments (●) ACE polysaccharides: A. camphorata extract, polysaccharides; (○) ACE/CS: ACE polysaccharides encapsulated by chitosan-silica nanoparticles; (▼) ACE/S: ACE polysaccharides encapsulated by silica nanoparticles; (■) SNP: silica nanoparticles; (□) CSNP: chitosan-silica nanoparticles.
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pone.0136782.g001: The cytotoxicity of ACE polysaccharides, ACE/CS and ACE/S on Hep G2 cells.Cells were incubated with samples for (a) 24 h or (b) 48 h and viability was assessed using an MTT assay. Nanoparticles without AEC polysaccharides were presented as (c) for 48 h incubation. Experiments were repeated 3 times independently to ensure reproducibility and the standard deviation of the mean are represented as error bars (n = 3). Values with different letters or asterisks were significantly different (p < 0.05) at corresponding concentrations between different treatments (●) ACE polysaccharides: A. camphorata extract, polysaccharides; (○) ACE/CS: ACE polysaccharides encapsulated by chitosan-silica nanoparticles; (▼) ACE/S: ACE polysaccharides encapsulated by silica nanoparticles; (■) SNP: silica nanoparticles; (□) CSNP: chitosan-silica nanoparticles.

Mentions: The death of Hep G2 cells was induced by ACE polysaccharides, ACE/CS and ACE/S in a dose and time-dependent manner (Fig 1). The results showed a slight decrease in cell viability at 24 h but became very significant after 48 h incubation, especially in the ACE/CS group (Fig 1b). Meanwhile, the viability of Hep G2 cells only decreased to approximately 85% in chitosan-silica nanoparticle (CSNP) or silica nanoparticle (SNP) treatments, which without ACE polysaccharides were loaded even at the highest concentration of 667 μg/mL for 48 h incubation (Fig 1c). Regarding morphology, cell shrinkage was obviously observed in phase-contract micrographs when exposed to ACE/CS (Fig 2a–2d) and a positive control (Fig 2e), but was insignificant in the CSNP treatment (Fig 2g). The effects of ACE polysaccharides and synthesized nanoparticles on Hep G2 cell membrane integrity, determined by a LDH assay, are shown in Fig 3a–3c. ACE polysaccharides, ACE/CS and ACE/S caused cell membrane damage in a dose- and time-dependent manner, especially at the highest concentration of 166.67 μg/mL ACE polysaccharide treatments for 48 h (> 60% LDH released). At the corresponding concentration; e.g., 166.67 μg/mL, ACE/ES and ACE/S (containing 13.2 μg/mL and 21.2 μg/mL ACE polysaccharides, respectively) showed much less membrane damage (< 20% LDH released; Fig 3b and 3c). The effects of nanoparticles without ACE polysaccharides on Hep G2 cell membrane integrity are shown in Fig 3d. None of the chitosan-silica composite treatments made a significant membranolytic effect up to 48 h even at a concentration of 667 μg/mL (< 3.7% LDH released). In contrast, silica nanoparticles were found to cause slight (< 6.5% LDH released) membrane damage in the same condition.


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 cytotoxicity of ACE polysaccharides, ACE/CS and ACE/S on Hep G2 cells.Cells were incubated with samples for (a) 24 h or (b) 48 h and viability was assessed using an MTT assay. Nanoparticles without AEC polysaccharides were presented as (c) for 48 h incubation. Experiments were repeated 3 times independently to ensure reproducibility and the standard deviation of the mean are represented as error bars (n = 3). Values with different letters or asterisks were significantly different (p < 0.05) at corresponding concentrations between different treatments (●) ACE polysaccharides: A. camphorata extract, polysaccharides; (○) ACE/CS: ACE polysaccharides encapsulated by chitosan-silica nanoparticles; (▼) ACE/S: ACE polysaccharides encapsulated by silica nanoparticles; (■) SNP: silica nanoparticles; (□) CSNP: chitosan-silica nanoparticles.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556685&req=5

pone.0136782.g001: The cytotoxicity of ACE polysaccharides, ACE/CS and ACE/S on Hep G2 cells.Cells were incubated with samples for (a) 24 h or (b) 48 h and viability was assessed using an MTT assay. Nanoparticles without AEC polysaccharides were presented as (c) for 48 h incubation. Experiments were repeated 3 times independently to ensure reproducibility and the standard deviation of the mean are represented as error bars (n = 3). Values with different letters or asterisks were significantly different (p < 0.05) at corresponding concentrations between different treatments (●) ACE polysaccharides: A. camphorata extract, polysaccharides; (○) ACE/CS: ACE polysaccharides encapsulated by chitosan-silica nanoparticles; (▼) ACE/S: ACE polysaccharides encapsulated by silica nanoparticles; (■) SNP: silica nanoparticles; (□) CSNP: chitosan-silica nanoparticles.
Mentions: The death of Hep G2 cells was induced by ACE polysaccharides, ACE/CS and ACE/S in a dose and time-dependent manner (Fig 1). The results showed a slight decrease in cell viability at 24 h but became very significant after 48 h incubation, especially in the ACE/CS group (Fig 1b). Meanwhile, the viability of Hep G2 cells only decreased to approximately 85% in chitosan-silica nanoparticle (CSNP) or silica nanoparticle (SNP) treatments, which without ACE polysaccharides were loaded even at the highest concentration of 667 μg/mL for 48 h incubation (Fig 1c). Regarding morphology, cell shrinkage was obviously observed in phase-contract micrographs when exposed to ACE/CS (Fig 2a–2d) and a positive control (Fig 2e), but was insignificant in the CSNP treatment (Fig 2g). The effects of ACE polysaccharides and synthesized nanoparticles on Hep G2 cell membrane integrity, determined by a LDH assay, are shown in Fig 3a–3c. ACE polysaccharides, ACE/CS and ACE/S caused cell membrane damage in a dose- and time-dependent manner, especially at the highest concentration of 166.67 μg/mL ACE polysaccharide treatments for 48 h (> 60% LDH released). At the corresponding concentration; e.g., 166.67 μg/mL, ACE/ES and ACE/S (containing 13.2 μg/mL and 21.2 μg/mL ACE polysaccharides, respectively) showed much less membrane damage (< 20% LDH released; Fig 3b and 3c). The effects of nanoparticles without ACE polysaccharides on Hep G2 cell membrane integrity are shown in Fig 3d. None of the chitosan-silica composite treatments made a significant membranolytic effect up to 48 h even at a concentration of 667 μg/mL (< 3.7% LDH released). In contrast, silica nanoparticles were found to cause slight (< 6.5% LDH released) membrane damage in the same condition.

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