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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

Gel electrophoresis of DNA genomes extracted from various treated Chang's and untreated HepG2 cells. Cells were incubated without or with various concentrations of ethanolic extract for 72 hours. The DNA fragments were separated using 1.5% agarose gel electrophoresis and visualised under UV light after staining with ethidium bromide. M: 100 bp DNA ladder marker, lane 1: non-malignant Chang's liver treated with 10 μg mL-1 of P. sarmentosum ethanolic extract, lane 2: non-malignant Chang's liver treated with 12 μg mL-1 of P. sarmentosum ethanolic extract, lane 3: non-malignant Chang's liver treated with 14 μg mL-1 of P. sarmentosum ethanolic extract, lane 4: untreated Chang's liver cells and lane 5: untreated HepG2 cell line. Each experiment was performed in triplicate (n = 3).
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Figure 8: Gel electrophoresis of DNA genomes extracted from various treated Chang's and untreated HepG2 cells. Cells were incubated without or with various concentrations of ethanolic extract for 72 hours. The DNA fragments were separated using 1.5% agarose gel electrophoresis and visualised under UV light after staining with ethidium bromide. M: 100 bp DNA ladder marker, lane 1: non-malignant Chang's liver treated with 10 μg mL-1 of P. sarmentosum ethanolic extract, lane 2: non-malignant Chang's liver treated with 12 μg mL-1 of P. sarmentosum ethanolic extract, lane 3: non-malignant Chang's liver treated with 14 μg mL-1 of P. sarmentosum ethanolic extract, lane 4: untreated Chang's liver cells and lane 5: untreated HepG2 cell line. Each experiment was performed in triplicate (n = 3).

Mentions: DNA fragmentation occurs in cells that produce intrinsic apoptosis activity when induced by a variety of agents. This cleavage produces ladders of DNA fragments that are the size of integer multiples of a nucleosome length (180–200 bp) [23]. The DNA fragmentation is initiated by caspase 3 activation of inactive CAD (caspase activated deoxyribonuclease) through removal of its inhibitors, i.e., ICAD [6]. As a biochemical hallmark of intrinsic apoptotic cell death, DNA fragmentation was used to determine whether the antiproliferative effect of P. sarmentosum ethanolic extract on cells acts through the respective apoptosis pathway [24]. As shown in Figure 7, the treatment of HepG2 cells with ethanolic extract resulted in the induction of intrinsic apoptosis activity at concentrations as low as 10 μg mL-1. HepG2 cells were treated with three different concentrations of ethanolic extract (10, 12 and 14 μg mL-1) based on the IC50 that was predetermined by MTT assay. HepG2 cells treated with different concentrations of ethanolic extract (Lane 1–3; Figure 7) for 72 hours showed typical features of DNA laddering on an agarose gel, whereas untreated cells produced intact genomes (Lane 5; Figure 8). In contrast, the non-malignant Chang's liver cell line when treated with the various concentrations of ethanolic extract (Lane 1–3; Figure 8) produced similar genomic DNA features as in untreated non-malignant Chang's liver (Lane 4; Figure 8) and HepG2 (Lane 5; Figure 8) cell lines. Therefore, the ethanolic extract at a concentration as low as 10 μg mL-1 can induce nucleosomal DNA fragmentation of HepG2 due to intrinsic apoptosis processes, but not in the non-malignant Chang's liver cell line. In this study, the reason of using "HepG2" and "Chang" liver cells because HepG2 are the model of hepatocellular carcinoma while Chang liver cells are considered as an in vitro model of non-malignant or non-tumor liver cells. This is based on other studies such as Antonin et al. and Teck et al. stated that Chang as non-malignant cells [25,26].


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)

Gel electrophoresis of DNA genomes extracted from various treated Chang's and untreated HepG2 cells. Cells were incubated without or with various concentrations of ethanolic extract for 72 hours. The DNA fragments were separated using 1.5% agarose gel electrophoresis and visualised under UV light after staining with ethidium bromide. M: 100 bp DNA ladder marker, lane 1: non-malignant Chang's liver treated with 10 μg mL-1 of P. sarmentosum ethanolic extract, lane 2: non-malignant Chang's liver treated with 12 μg mL-1 of P. sarmentosum ethanolic extract, lane 3: non-malignant Chang's liver treated with 14 μg mL-1 of P. sarmentosum ethanolic extract, lane 4: untreated Chang's liver cells and lane 5: untreated HepG2 cell line. Each experiment was performed in triplicate (n = 3).
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Figure 8: Gel electrophoresis of DNA genomes extracted from various treated Chang's and untreated HepG2 cells. Cells were incubated without or with various concentrations of ethanolic extract for 72 hours. The DNA fragments were separated using 1.5% agarose gel electrophoresis and visualised under UV light after staining with ethidium bromide. M: 100 bp DNA ladder marker, lane 1: non-malignant Chang's liver treated with 10 μg mL-1 of P. sarmentosum ethanolic extract, lane 2: non-malignant Chang's liver treated with 12 μg mL-1 of P. sarmentosum ethanolic extract, lane 3: non-malignant Chang's liver treated with 14 μg mL-1 of P. sarmentosum ethanolic extract, lane 4: untreated Chang's liver cells and lane 5: untreated HepG2 cell line. Each experiment was performed in triplicate (n = 3).
Mentions: DNA fragmentation occurs in cells that produce intrinsic apoptosis activity when induced by a variety of agents. This cleavage produces ladders of DNA fragments that are the size of integer multiples of a nucleosome length (180–200 bp) [23]. The DNA fragmentation is initiated by caspase 3 activation of inactive CAD (caspase activated deoxyribonuclease) through removal of its inhibitors, i.e., ICAD [6]. As a biochemical hallmark of intrinsic apoptotic cell death, DNA fragmentation was used to determine whether the antiproliferative effect of P. sarmentosum ethanolic extract on cells acts through the respective apoptosis pathway [24]. As shown in Figure 7, the treatment of HepG2 cells with ethanolic extract resulted in the induction of intrinsic apoptosis activity at concentrations as low as 10 μg mL-1. HepG2 cells were treated with three different concentrations of ethanolic extract (10, 12 and 14 μg mL-1) based on the IC50 that was predetermined by MTT assay. HepG2 cells treated with different concentrations of ethanolic extract (Lane 1–3; Figure 7) for 72 hours showed typical features of DNA laddering on an agarose gel, whereas untreated cells produced intact genomes (Lane 5; Figure 8). In contrast, the non-malignant Chang's liver cell line when treated with the various concentrations of ethanolic extract (Lane 1–3; Figure 8) produced similar genomic DNA features as in untreated non-malignant Chang's liver (Lane 4; Figure 8) and HepG2 (Lane 5; Figure 8) cell lines. Therefore, the ethanolic extract at a concentration as low as 10 μg mL-1 can induce nucleosomal DNA fragmentation of HepG2 due to intrinsic apoptosis processes, but not in the non-malignant Chang's liver cell line. In this study, the reason of using "HepG2" and "Chang" liver cells because HepG2 are the model of hepatocellular carcinoma while Chang liver cells are considered as an in vitro model of non-malignant or non-tumor liver cells. This is based on other studies such as Antonin et al. and Teck et al. stated that Chang as non-malignant cells [25,26].

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