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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

Percentages of HepG2 cell death via apoptosis after treatment with ethanolic extract from P. sarmentosum. The percentages of HepG2 cell death via apoptosis increased significantly in a time-dependent manner. p < 0.05 represents the statistically significant difference between the control and treated group (1% DMSO, 24–72 hours of incubation in 12.5 μg mL-1 of ethanolic extract). *Represents significant results (p < 0.05) using ANOVA statistical analysis when the treated group was compared with the control.
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Figure 5: Percentages of HepG2 cell death via apoptosis after treatment with ethanolic extract from P. sarmentosum. The percentages of HepG2 cell death via apoptosis increased significantly in a time-dependent manner. p < 0.05 represents the statistically significant difference between the control and treated group (1% DMSO, 24–72 hours of incubation in 12.5 μg mL-1 of ethanolic extract). *Represents significant results (p < 0.05) using ANOVA statistical analysis when the treated group was compared with the control.

Mentions: The apoptotic index (AI) was calculated to confirm that ethanolic-treated cell death was via apoptosis. AI is described as the percentage of apoptotic cells and apoptotic bodies within the overall population of cells [21]. An apoptotic index was determined as the percentage of apoptotic cells from at least 400 counted cells under observation using an inverted microscope. The statistical differences between the control group and treated group (1% DMSO, 24 hours, 48 hours and 72 hours) were analysed using ANOVA, and p values less than 0.05 were considered as significant. The percentages of apoptotic cells after treatment were increased in a time-dependent manner with less than 50% at 24 hours, more than 50% at 48 hours and even higher at 72 hours. Untreated cells are represented as the control, i.e., the HepG2 cell line cultured in complete media for 72 hours. The control cells showed that only 4% of these cell deaths produced a typical morphological apoptotic feature (Figure 5). On the other hand, cells treated with 1% DMSO (negative control) for 72 hours produced only 8% cell death and showed no significant difference (p > 0.05) when compared to untreated cells (control). In contrast, Figure 5 also showed that the AI percentage of HepG2 cells increased significantly (p < 0.05) when the HepG2 cell line was treated with 12.5 μg mL-1 of ethanolic extract from P. sarmentosum at 24 hours compared to the control. The AI percentage of the HepG2 cell line also continued to increase significantly (p < 0.05) when the HepG2 cell line was treated with ethanolic extracts at 48 and 72 hours compared to the control (Figure 5). This observation indicated that the apoptotic activity was gradually increased when the ethanolic extract was incubated longer in carcinoma HepG2 cells.


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)

Percentages of HepG2 cell death via apoptosis after treatment with ethanolic extract from P. sarmentosum. The percentages of HepG2 cell death via apoptosis increased significantly in a time-dependent manner. p < 0.05 represents the statistically significant difference between the control and treated group (1% DMSO, 24–72 hours of incubation in 12.5 μg mL-1 of ethanolic extract). *Represents significant results (p < 0.05) using ANOVA statistical analysis when the treated group was compared with the control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Percentages of HepG2 cell death via apoptosis after treatment with ethanolic extract from P. sarmentosum. The percentages of HepG2 cell death via apoptosis increased significantly in a time-dependent manner. p < 0.05 represents the statistically significant difference between the control and treated group (1% DMSO, 24–72 hours of incubation in 12.5 μg mL-1 of ethanolic extract). *Represents significant results (p < 0.05) using ANOVA statistical analysis when the treated group was compared with the control.
Mentions: The apoptotic index (AI) was calculated to confirm that ethanolic-treated cell death was via apoptosis. AI is described as the percentage of apoptotic cells and apoptotic bodies within the overall population of cells [21]. An apoptotic index was determined as the percentage of apoptotic cells from at least 400 counted cells under observation using an inverted microscope. The statistical differences between the control group and treated group (1% DMSO, 24 hours, 48 hours and 72 hours) were analysed using ANOVA, and p values less than 0.05 were considered as significant. The percentages of apoptotic cells after treatment were increased in a time-dependent manner with less than 50% at 24 hours, more than 50% at 48 hours and even higher at 72 hours. Untreated cells are represented as the control, i.e., the HepG2 cell line cultured in complete media for 72 hours. The control cells showed that only 4% of these cell deaths produced a typical morphological apoptotic feature (Figure 5). On the other hand, cells treated with 1% DMSO (negative control) for 72 hours produced only 8% cell death and showed no significant difference (p > 0.05) when compared to untreated cells (control). In contrast, Figure 5 also showed that the AI percentage of HepG2 cells increased significantly (p < 0.05) when the HepG2 cell line was treated with 12.5 μg mL-1 of ethanolic extract from P. sarmentosum at 24 hours compared to the control. The AI percentage of the HepG2 cell line also continued to increase significantly (p < 0.05) when the HepG2 cell line was treated with ethanolic extracts at 48 and 72 hours compared to the control (Figure 5). This observation indicated that the apoptotic activity was gradually increased when the ethanolic extract was incubated longer in carcinoma HepG2 cells.

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