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Intrinsic anticarcinogenic effects of Piper sarmentosum ethanolic extract on a human hepatoma cell line.

Zainal Ariffin SH, Wan Omar WH, Zainal Ariffin Z, Safian MF, Senafi S, Megat Abdul Wahab R - Cancer Cell Int. (2009)

Bottom Line: The percentage of apoptotic cells in the overall population (apoptotic index) showed a continuously significant increase (p < 0.05) in 12.5 mug mL-1 ethanolic extract-treated cells at 24, 48 and 72 hours compared to controls (untreated cells).These results showed a typical intrinsic apoptotic characterisation, which included fragmentation of nuclear DNA in ethanolic extract-treated HepG2 cells.Therefore, our results suggest that the ethanolic extract from P. sarmentosum induced anticarcinogenic activity through an intrinsic apoptosis pathway in HepG2 cells in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Selangor Darul Ehsan, Malaysia. shahroy8@gmail.com

ABSTRACT

Background: Piper sarmentosum, locally known as kaduk is belonging to the family of Piperaceae. It is our interest to evaluate their effect on human hepatoma cell line (HepG2) for the potential of anticarcinogenic activity.

Results: The anticarcinogenic activity of an ethanolic extract from Piper sarmentosum in HepG2 and non-malignant Chang's liver cell lines has been previously determined using (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) (MTT) assays, where the IC50 value was used as a parameter for cytotoxicity. The ethanolic extract that showed anticarcinogenic properties in HepG2 cells had an IC50 of 12.5 mug mL-1, while IC50 values in the non-malignant Chang's liver cell line were greater than 30 mug mL-1. Apoptotic morphological changes in HepG2 cells were observed using an inverted microscope and showed chromatin condensation, cell shrinkage and apoptotic bodies following May-Grunwald-Giemsa's staining. The percentage of apoptotic cells in the overall population (apoptotic index) showed a continuously significant increase (p < 0.05) in 12.5 mug mL-1 ethanolic extract-treated cells at 24, 48 and 72 hours compared to controls (untreated cells). Following acridine orange and ethidium bromide staining, treatment with 10, 12 and 14 mug mL-1 of ethanolic extracts caused typical apoptotic morphological changes in HepG2 cells. Molecular analysis of DNA fragmentation was used to examine intrinsic apoptosis induced by the ethanolic extracts. These results showed a typical intrinsic apoptotic characterisation, which included fragmentation of nuclear DNA in ethanolic extract-treated HepG2 cells. However, the non-malignant Chang's liver cell line produced no DNA fragmentation. In addition, the DNA genome was similarly intact for both the untreated non-malignant Chang's liver and HepG2 cell lines.

Conclusion: Therefore, our results suggest that the ethanolic extract from P. sarmentosum induced anticarcinogenic activity through an intrinsic apoptosis pathway in HepG2 cells in vitro.

No MeSH data available.


Related in: MedlinePlus

Morphological observation with acridine orange and ethidium bromide (AO/EB) staining at actual magnification 400×. HepG2 cells were treated without (A) and with P. sarmentosum ethanolic extract, 10 μg mL-1 (B), 12 μg mL-1 (C) and 14 μg mL-1 (D) for 72 hours. Dashed arrow indicated cells with chromatin condensation; rounded dotted arrow indicated cells with fragmented nuclei; dashed dotted arrow indicated cells with membrane blebbing and full white arrow indicated the presence of apoptotic bodies. Each experiment was performed in triplicate (n = 3) and generated similar morphological features.
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Figure 6: Morphological observation with acridine orange and ethidium bromide (AO/EB) staining at actual magnification 400×. HepG2 cells were treated without (A) and with P. sarmentosum ethanolic extract, 10 μg mL-1 (B), 12 μg mL-1 (C) and 14 μg mL-1 (D) for 72 hours. Dashed arrow indicated cells with chromatin condensation; rounded dotted arrow indicated cells with fragmented nuclei; dashed dotted arrow indicated cells with membrane blebbing and full white arrow indicated the presence of apoptotic bodies. Each experiment was performed in triplicate (n = 3) and generated similar morphological features.

Mentions: Staining cells with fluorescent dyes, including acridine orange and ethidium bromide, is used in evaluating the nuclear morphology of apoptotic cells. To corroborate that apoptosis has been induced by P. sarmentosum ethanolic plant extract, HepG2 cells were analysed in the presence of acridine orange and ethidium bromide staining (AO/EB staining). Acridine orange is a vital dye that will stain both live and dead cells, whereas ethidium bromide will stain only those cells that have lost their membrane integrity [22]. Three different concentrations were chosen based on the IC50 values determined by MTT assay, which were 10, 12 and 14 μg mL-1. As a control, HepG2 cells were cultured in complete media and stained with AO/EB (Figure 6A). The figure shows that the ethanolic extract from P. sarmentosum induced apoptosis after 72 hours incubation at all concentrations of plant extract tested. Cells stained green represent viable cells, whereas yellow staining represented early apoptotic cells, and reddish or orange staining represents late apoptotic cells. As shown in Figure 6B, HepG2 cells treated with 10 μg mL-1 of ethanolic extract showed changes in cellular morphology, including chromatin condensation, membrane blebbing, and fragmented nuclei. On the other hand, Figures 6C and 6D show similar features for cells treated with 10 μg mL-1 of ethanolic extract (Figure 6B), but with extra features of late stage apoptotic activity with apoptotic bodies when HepG2 cells were treated with 12 μg mL-1 and 14 μg mL-1 of ethanolic extract from P. sarmentosum. Therefore, using the AO/EB staining procedure, the morphological features of a hepatoma cell line in apoptosis were dose dependent, i.e., a stronger apoptosis signal was induced with higher concentrations of the respective extract.


Intrinsic anticarcinogenic effects of Piper sarmentosum ethanolic extract on a human hepatoma cell line.

Zainal Ariffin SH, Wan Omar WH, Zainal Ariffin Z, Safian MF, Senafi S, Megat Abdul Wahab R - Cancer Cell Int. (2009)

Morphological observation with acridine orange and ethidium bromide (AO/EB) staining at actual magnification 400×. HepG2 cells were treated without (A) and with P. sarmentosum ethanolic extract, 10 μg mL-1 (B), 12 μg mL-1 (C) and 14 μg mL-1 (D) for 72 hours. Dashed arrow indicated cells with chromatin condensation; rounded dotted arrow indicated cells with fragmented nuclei; dashed dotted arrow indicated cells with membrane blebbing and full white arrow indicated the presence of apoptotic bodies. Each experiment was performed in triplicate (n = 3) and generated similar morphological features.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2667431&req=5

Figure 6: Morphological observation with acridine orange and ethidium bromide (AO/EB) staining at actual magnification 400×. HepG2 cells were treated without (A) and with P. sarmentosum ethanolic extract, 10 μg mL-1 (B), 12 μg mL-1 (C) and 14 μg mL-1 (D) for 72 hours. Dashed arrow indicated cells with chromatin condensation; rounded dotted arrow indicated cells with fragmented nuclei; dashed dotted arrow indicated cells with membrane blebbing and full white arrow indicated the presence of apoptotic bodies. Each experiment was performed in triplicate (n = 3) and generated similar morphological features.
Mentions: Staining cells with fluorescent dyes, including acridine orange and ethidium bromide, is used in evaluating the nuclear morphology of apoptotic cells. To corroborate that apoptosis has been induced by P. sarmentosum ethanolic plant extract, HepG2 cells were analysed in the presence of acridine orange and ethidium bromide staining (AO/EB staining). Acridine orange is a vital dye that will stain both live and dead cells, whereas ethidium bromide will stain only those cells that have lost their membrane integrity [22]. Three different concentrations were chosen based on the IC50 values determined by MTT assay, which were 10, 12 and 14 μg mL-1. As a control, HepG2 cells were cultured in complete media and stained with AO/EB (Figure 6A). The figure shows that the ethanolic extract from P. sarmentosum induced apoptosis after 72 hours incubation at all concentrations of plant extract tested. Cells stained green represent viable cells, whereas yellow staining represented early apoptotic cells, and reddish or orange staining represents late apoptotic cells. As shown in Figure 6B, HepG2 cells treated with 10 μg mL-1 of ethanolic extract showed changes in cellular morphology, including chromatin condensation, membrane blebbing, and fragmented nuclei. On the other hand, Figures 6C and 6D show similar features for cells treated with 10 μg mL-1 of ethanolic extract (Figure 6B), but with extra features of late stage apoptotic activity with apoptotic bodies when HepG2 cells were treated with 12 μg mL-1 and 14 μg mL-1 of ethanolic extract from P. sarmentosum. Therefore, using the AO/EB staining procedure, the morphological features of a hepatoma cell line in apoptosis were dose dependent, i.e., a stronger apoptosis signal was induced with higher concentrations of the respective extract.

Bottom Line: The percentage of apoptotic cells in the overall population (apoptotic index) showed a continuously significant increase (p < 0.05) in 12.5 mug mL-1 ethanolic extract-treated cells at 24, 48 and 72 hours compared to controls (untreated cells).These results showed a typical intrinsic apoptotic characterisation, which included fragmentation of nuclear DNA in ethanolic extract-treated HepG2 cells.Therefore, our results suggest that the ethanolic extract from P. sarmentosum induced anticarcinogenic activity through an intrinsic apoptosis pathway in HepG2 cells in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Selangor Darul Ehsan, Malaysia. shahroy8@gmail.com

ABSTRACT

Background: Piper sarmentosum, locally known as kaduk is belonging to the family of Piperaceae. It is our interest to evaluate their effect on human hepatoma cell line (HepG2) for the potential of anticarcinogenic activity.

Results: The anticarcinogenic activity of an ethanolic extract from Piper sarmentosum in HepG2 and non-malignant Chang's liver cell lines has been previously determined using (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) (MTT) assays, where the IC50 value was used as a parameter for cytotoxicity. The ethanolic extract that showed anticarcinogenic properties in HepG2 cells had an IC50 of 12.5 mug mL-1, while IC50 values in the non-malignant Chang's liver cell line were greater than 30 mug mL-1. Apoptotic morphological changes in HepG2 cells were observed using an inverted microscope and showed chromatin condensation, cell shrinkage and apoptotic bodies following May-Grunwald-Giemsa's staining. The percentage of apoptotic cells in the overall population (apoptotic index) showed a continuously significant increase (p < 0.05) in 12.5 mug mL-1 ethanolic extract-treated cells at 24, 48 and 72 hours compared to controls (untreated cells). Following acridine orange and ethidium bromide staining, treatment with 10, 12 and 14 mug mL-1 of ethanolic extracts caused typical apoptotic morphological changes in HepG2 cells. Molecular analysis of DNA fragmentation was used to examine intrinsic apoptosis induced by the ethanolic extracts. These results showed a typical intrinsic apoptotic characterisation, which included fragmentation of nuclear DNA in ethanolic extract-treated HepG2 cells. However, the non-malignant Chang's liver cell line produced no DNA fragmentation. In addition, the DNA genome was similarly intact for both the untreated non-malignant Chang's liver and HepG2 cell lines.

Conclusion: Therefore, our results suggest that the ethanolic extract from P. sarmentosum induced anticarcinogenic activity through an intrinsic apoptosis pathway in HepG2 cells in vitro.

No MeSH data available.


Related in: MedlinePlus