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Pleiocarpa pycnantha leaves and its triterpenes induce apoptotic cell death in Caco-2 cells in vitro.

Omoyeni OA, Hussein A, Meyer M, Green I, Iwuoha E - BMC Complement Altern Med (2015)

Bottom Line: The exposure of an ethanolic extract from the leaves of P. pycnantha (0.1-1000 μg/ml) and the isolated compounds C2 and C3 (6,25-100 μg/ml) to human colorectal cancer cells reduced the cell viability with an IC50 > 100, 40.9, 36.3 μg/ml for P, C2 and C3 respectively, after 24 h of incubation.Caspase 3 was also activated which is a hallmark of apoptosis.A further study with other cell lines is also recommended.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of the Western Cape, Bellville, South Africa. nikxyglo@yahoo.com.

ABSTRACT

Background: Recently, we reported that the crude fractions and pure triterpenes; ursolic acid (C1), 27-E and 27-Z p-coumaric esters of ursolic acid (C2, C3), together with a new triterpene 2,3-seco-taraxer-14-en-2,3-lactone [pycanocarpine (C4)] and its hydrolysed derivative - (2,3-seco-taraxen-4-hydroxy-14-en-2-oic acid) [pycanocarpene (C5)] from Pleiocarpa pycnantha leaves inhibit cell proliferation. However, there has not been any specific report on the use of Pleiocarpa pycnantha leaves and its constituents to kill colorectal adenocarcinoma cancer CaCo-2 cells. We performed in vitro study to evaluate the cytotoxic properties of the ethanolic extract of P. pycnantha P, compounds C2 and C3. A preliminary study of the potential mechanisms were also undertaken.

Methods: Cell viability was measured by WST-1 assay. The Apoptosis level was evaluated by staining with APOPercentage(™) dye and the induction of caspases 3/7 and 9 using Caspase-Glo(®) assays.

Results: The exposure of an ethanolic extract from the leaves of P. pycnantha (0.1-1000 μg/ml) and the isolated compounds C2 and C3 (6,25-100 μg/ml) to human colorectal cancer cells reduced the cell viability with an IC50 > 100, 40.9, 36.3 μg/ml for P, C2 and C3 respectively, after 24 h of incubation. The APOPercentage(TM) assay also showed a considerable increase in the percentage of apoptotic cells after 24 h; (25-38% for P, 5-23% for C2 and 6-47 % for C3). Caspase 3 was also activated which is a hallmark of apoptosis.

Conclusion: These findings suggest that the P. pycnantha and the isolated compounds induce cell apoptosis in human colorectal adenocarcinoma cells. A further study with other cell lines is also recommended.

No MeSH data available.


Related in: MedlinePlus

Pleiocarpa pycnantha and isolated compounds apoptosis is mediated by caspase3/7 activation. a Treatment of cells with various concentration of extract P for 6–24 h (P) treated with various concentrations for 24 h. b Cells were treated with increasing concentrations of compound C2 for 6–24 h. c Measurement of caspase 3/7 activity when cells were treated with different concentrations of compound C3 for 6–24 h. Data are presented as mean ± SEM,***P < 0.001, **P < 0.01 and *P < 0.05 compared with control
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Fig4: Pleiocarpa pycnantha and isolated compounds apoptosis is mediated by caspase3/7 activation. a Treatment of cells with various concentration of extract P for 6–24 h (P) treated with various concentrations for 24 h. b Cells were treated with increasing concentrations of compound C2 for 6–24 h. c Measurement of caspase 3/7 activity when cells were treated with different concentrations of compound C3 for 6–24 h. Data are presented as mean ± SEM,***P < 0.001, **P < 0.01 and *P < 0.05 compared with control

Mentions: The activation of caspases, which are mediators of apoptosis was analysed upon exposure of Caco-2 cells to the P. pycnantha ethanol extract and the compounds C2 and C3. The expression of caspase 3/7 and 9 activity was measured in cells exposed to several concentrations of the extract (10–100 μg/ml) for 6–24 h or incubated with C2 and C3 (12.5–25 μg/ml) for 6–24 h. The levels of caspase activation in Caco-2 cells were compared with untreated control cells arbitrarily set to 1.0, the results showed that the extract P significantly increase caspase 3/7 activation at different concentrations and time. At P10 μg/ml, the caspase 3/7 activity reached a maximum (1.62 fold increase at 24 h (Fig.4a). A similar occurrence was observed at P100 μg/ml (1.35 fold increase), while at P50 μg/ml, the maximum caspase activity was recorded at 12 h with approximate fold increase being 1.49 (Fig.4a). Furthermore, compound C2 at 12.5 μg/ml showed a significant caspase 3/7 ≈ 2.0 fold increase activity as early as 6 h while C3 at the same dose had about 1.1 fold increase (Fig.4b and c). At 12 h, compound C3 (12.5 μg/ml) also showed a marked increase caspase activity (1.76 fold increase) as compared with the control, C2 at a similar dose showed only 1.2 fold increase when compared with the control. A similar trend was also observed at 24 h of treatment. Compounds C2 and C3 did not show any significant effect on caspase activation at a higher dose of 25 μg/ml at 6–24 h of treatment (Fig.4b and c). The result of caspase 9 activity of P, C2 and C3 on Caco-2 cells are shown in Fig.5a-c. The result further showed that at concentrations 10–100 μg/ml, P significantly activated caspase 9 at different time points as compared with the untreated control except at 6 h for P10 μg/ml (Fig. 5a). At concentrations, 10, 50, and 100 μg/ml, the maximum caspase 9 activity fold increase obtained were 1.69, 1.79 and 1.45 at 24 h and 12 h respectively. At 25 μg/ml, C2 showed a significant increased caspase 9 activity within 6–24 h of treatment with 1.34–1.41 fold increase (Fig. 5b). Similarly, compound C3 at 25 μg/ml showed an increased caspase 9 activity when compared with the untreated control within 6–24 h from 1.05–1.27 (Fig. 5c). Only a slight increase was observed at the concentration12.5 μg/ml for both C2 and C3 at 6–24 h.Fig. 4


Pleiocarpa pycnantha leaves and its triterpenes induce apoptotic cell death in Caco-2 cells in vitro.

Omoyeni OA, Hussein A, Meyer M, Green I, Iwuoha E - BMC Complement Altern Med (2015)

Pleiocarpa pycnantha and isolated compounds apoptosis is mediated by caspase3/7 activation. a Treatment of cells with various concentration of extract P for 6–24 h (P) treated with various concentrations for 24 h. b Cells were treated with increasing concentrations of compound C2 for 6–24 h. c Measurement of caspase 3/7 activity when cells were treated with different concentrations of compound C3 for 6–24 h. Data are presented as mean ± SEM,***P < 0.001, **P < 0.01 and *P < 0.05 compared with control
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4499947&req=5

Fig4: Pleiocarpa pycnantha and isolated compounds apoptosis is mediated by caspase3/7 activation. a Treatment of cells with various concentration of extract P for 6–24 h (P) treated with various concentrations for 24 h. b Cells were treated with increasing concentrations of compound C2 for 6–24 h. c Measurement of caspase 3/7 activity when cells were treated with different concentrations of compound C3 for 6–24 h. Data are presented as mean ± SEM,***P < 0.001, **P < 0.01 and *P < 0.05 compared with control
Mentions: The activation of caspases, which are mediators of apoptosis was analysed upon exposure of Caco-2 cells to the P. pycnantha ethanol extract and the compounds C2 and C3. The expression of caspase 3/7 and 9 activity was measured in cells exposed to several concentrations of the extract (10–100 μg/ml) for 6–24 h or incubated with C2 and C3 (12.5–25 μg/ml) for 6–24 h. The levels of caspase activation in Caco-2 cells were compared with untreated control cells arbitrarily set to 1.0, the results showed that the extract P significantly increase caspase 3/7 activation at different concentrations and time. At P10 μg/ml, the caspase 3/7 activity reached a maximum (1.62 fold increase at 24 h (Fig.4a). A similar occurrence was observed at P100 μg/ml (1.35 fold increase), while at P50 μg/ml, the maximum caspase activity was recorded at 12 h with approximate fold increase being 1.49 (Fig.4a). Furthermore, compound C2 at 12.5 μg/ml showed a significant caspase 3/7 ≈ 2.0 fold increase activity as early as 6 h while C3 at the same dose had about 1.1 fold increase (Fig.4b and c). At 12 h, compound C3 (12.5 μg/ml) also showed a marked increase caspase activity (1.76 fold increase) as compared with the control, C2 at a similar dose showed only 1.2 fold increase when compared with the control. A similar trend was also observed at 24 h of treatment. Compounds C2 and C3 did not show any significant effect on caspase activation at a higher dose of 25 μg/ml at 6–24 h of treatment (Fig.4b and c). The result of caspase 9 activity of P, C2 and C3 on Caco-2 cells are shown in Fig.5a-c. The result further showed that at concentrations 10–100 μg/ml, P significantly activated caspase 9 at different time points as compared with the untreated control except at 6 h for P10 μg/ml (Fig. 5a). At concentrations, 10, 50, and 100 μg/ml, the maximum caspase 9 activity fold increase obtained were 1.69, 1.79 and 1.45 at 24 h and 12 h respectively. At 25 μg/ml, C2 showed a significant increased caspase 9 activity within 6–24 h of treatment with 1.34–1.41 fold increase (Fig. 5b). Similarly, compound C3 at 25 μg/ml showed an increased caspase 9 activity when compared with the untreated control within 6–24 h from 1.05–1.27 (Fig. 5c). Only a slight increase was observed at the concentration12.5 μg/ml for both C2 and C3 at 6–24 h.Fig. 4

Bottom Line: The exposure of an ethanolic extract from the leaves of P. pycnantha (0.1-1000 μg/ml) and the isolated compounds C2 and C3 (6,25-100 μg/ml) to human colorectal cancer cells reduced the cell viability with an IC50 > 100, 40.9, 36.3 μg/ml for P, C2 and C3 respectively, after 24 h of incubation.Caspase 3 was also activated which is a hallmark of apoptosis.A further study with other cell lines is also recommended.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of the Western Cape, Bellville, South Africa. nikxyglo@yahoo.com.

ABSTRACT

Background: Recently, we reported that the crude fractions and pure triterpenes; ursolic acid (C1), 27-E and 27-Z p-coumaric esters of ursolic acid (C2, C3), together with a new triterpene 2,3-seco-taraxer-14-en-2,3-lactone [pycanocarpine (C4)] and its hydrolysed derivative - (2,3-seco-taraxen-4-hydroxy-14-en-2-oic acid) [pycanocarpene (C5)] from Pleiocarpa pycnantha leaves inhibit cell proliferation. However, there has not been any specific report on the use of Pleiocarpa pycnantha leaves and its constituents to kill colorectal adenocarcinoma cancer CaCo-2 cells. We performed in vitro study to evaluate the cytotoxic properties of the ethanolic extract of P. pycnantha P, compounds C2 and C3. A preliminary study of the potential mechanisms were also undertaken.

Methods: Cell viability was measured by WST-1 assay. The Apoptosis level was evaluated by staining with APOPercentage(™) dye and the induction of caspases 3/7 and 9 using Caspase-Glo(®) assays.

Results: The exposure of an ethanolic extract from the leaves of P. pycnantha (0.1-1000 μg/ml) and the isolated compounds C2 and C3 (6,25-100 μg/ml) to human colorectal cancer cells reduced the cell viability with an IC50 > 100, 40.9, 36.3 μg/ml for P, C2 and C3 respectively, after 24 h of incubation. The APOPercentage(TM) assay also showed a considerable increase in the percentage of apoptotic cells after 24 h; (25-38% for P, 5-23% for C2 and 6-47 % for C3). Caspase 3 was also activated which is a hallmark of apoptosis.

Conclusion: These findings suggest that the P. pycnantha and the isolated compounds induce cell apoptosis in human colorectal adenocarcinoma cells. A further study with other cell lines is also recommended.

No MeSH data available.


Related in: MedlinePlus