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Liriodenine, an aporphine alkaloid from Enicosanthellum pulchrum, inhibits proliferation of human ovarian cancer cells through induction of apoptosis via the mitochondrial signaling pathway and blocking cell cycle progression.

Nordin N, Majid NA, Hashim NM, Rahman MA, Hassan Z, Ali HM - Drug Des Devel Ther (2015)

Bottom Line: The result showed that liriodenine inhibits proliferation of CAOV-3 cells at 37.3 μM after 24 hours of exposure.Involvement of the intrinsic pathway in the mitochondria could be seen, with a significant increase in mitochondrial permeability and cytochrome c release, whereas the mitochondrial membrane potential was decreased.These findings indicate that liriodenine could be considered as a promising anticancer agent.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.

ABSTRACT
Enicosanthellum pulchrum is a tropical plant from Malaysia and belongs to the Annonaceae family. This plant is rich in isoquinoline alkaloids. In the present study, liriodenine, an isoquinoline alkaloid, was examined as a potential anticancer agent, particularly in ovarian cancer. Liriodenine was isolated by preparative high-performance liquid chromatography. Cell viability was performed to determine the cytotoxicity, whilst the detection of morphological changes was carried out by acridine orange/propidium iodide assay. Initial and late apoptosis was examined by Annexin V-fluorescein isothiocyanate and DNA laddering assays, respectively. The involvement of pathways was detected via caspase-3, caspase-8, and caspase-9 analyses. Confirmation of pathways was further performed in mitochondria using a cytotoxicity 3 assay. Apoptosis was confirmed at the protein level, including Bax, Bcl-2, and survivin, while interruption of the cell cycle was used for final validation of apoptosis. The result showed that liriodenine inhibits proliferation of CAOV-3 cells at 37.3 μM after 24 hours of exposure. Changes in cell morphology were detected by the presence of cell membrane blebbing, chromatin condensation, and formation of apoptotic bodies. Early apoptosis was observed by Annexin V-fluorescein isothiocyanate bound to the cell membrane as early as 24 hours. Liriodenine activated the intrinsic pathway by induction of caspase-3 and caspase-9. Involvement of the intrinsic pathway in the mitochondria could be seen, with a significant increase in mitochondrial permeability and cytochrome c release, whereas the mitochondrial membrane potential was decreased. DNA fragmentation occurred at 72 hours upon exposure to liriodenine. The presence of DNA fragmentation indicates the CAOV-3 cells undergo late apoptosis or final stage of apoptosis. Confirmation of apoptosis at the protein level showed overexpression of Bax and suppression of Bcl-2 and survivin. Liriodenine inhibits progression of the CAOV-3 cell cycle in S phase. These findings indicate that liriodenine could be considered as a promising anticancer agent.

No MeSH data available.


Related in: MedlinePlus

Representative images and quantitative analysis of CAOV-3 cells treated or not treated with liriodenine at 24 hours.Notes: The cells were stained with Hoechst, fluorescein isothiocyanate, mitochondrial membrane potential, and cytochrome c dyes. The treated cells stained with Hoechst, fluorescein isothiocyanate, and cytochrome c dyes showed an increase in intensity that was dependent on the increase of liriodenine concentration, suggesting cell permeability was disrupted, allowing release of cytochrome c into the cytosol, while the mitochondrial membrane potential and cell numbers showed a reduction in fluorescence intensity. Average fluorescence intensities were observed simultaneously in CAOV-3 cells for all parameters. All data are shown as the mean ± standard deviation. Statistical significance was expressed at *P<0.05 (magnification 20×).
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f6-dddt-9-1437: Representative images and quantitative analysis of CAOV-3 cells treated or not treated with liriodenine at 24 hours.Notes: The cells were stained with Hoechst, fluorescein isothiocyanate, mitochondrial membrane potential, and cytochrome c dyes. The treated cells stained with Hoechst, fluorescein isothiocyanate, and cytochrome c dyes showed an increase in intensity that was dependent on the increase of liriodenine concentration, suggesting cell permeability was disrupted, allowing release of cytochrome c into the cytosol, while the mitochondrial membrane potential and cell numbers showed a reduction in fluorescence intensity. Average fluorescence intensities were observed simultaneously in CAOV-3 cells for all parameters. All data are shown as the mean ± standard deviation. Statistical significance was expressed at *P<0.05 (magnification 20×).

Mentions: The Multiparameter Cytotoxicity 3 assay (Cellomics) was carried out to investigate four parameters that are important in the apoptosis process, ie, total nuclear intensity, mitochondrial membrane potential, cell permeability, and cytochrome c release (Figure 6). The results indicated a reduction in total nuclei (Hoechst dye), which refers to the number of CAOV-3 cells present after being treated with liriodenine for 24 hours. The same situation occurred in the mitochondrial membrane potential, whereby there was a significant reduction in intensity (P<0.05) in cells treated with liriodenine 30 and 40 μM, respectively, at 24 hours. Meanwhile, cell permeability and cytochrome c were significantly increased (P<0.05) when compared with untreated CAOV-3 cells at the same concentrations.


Liriodenine, an aporphine alkaloid from Enicosanthellum pulchrum, inhibits proliferation of human ovarian cancer cells through induction of apoptosis via the mitochondrial signaling pathway and blocking cell cycle progression.

Nordin N, Majid NA, Hashim NM, Rahman MA, Hassan Z, Ali HM - Drug Des Devel Ther (2015)

Representative images and quantitative analysis of CAOV-3 cells treated or not treated with liriodenine at 24 hours.Notes: The cells were stained with Hoechst, fluorescein isothiocyanate, mitochondrial membrane potential, and cytochrome c dyes. The treated cells stained with Hoechst, fluorescein isothiocyanate, and cytochrome c dyes showed an increase in intensity that was dependent on the increase of liriodenine concentration, suggesting cell permeability was disrupted, allowing release of cytochrome c into the cytosol, while the mitochondrial membrane potential and cell numbers showed a reduction in fluorescence intensity. Average fluorescence intensities were observed simultaneously in CAOV-3 cells for all parameters. All data are shown as the mean ± standard deviation. Statistical significance was expressed at *P<0.05 (magnification 20×).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362660&req=5

f6-dddt-9-1437: Representative images and quantitative analysis of CAOV-3 cells treated or not treated with liriodenine at 24 hours.Notes: The cells were stained with Hoechst, fluorescein isothiocyanate, mitochondrial membrane potential, and cytochrome c dyes. The treated cells stained with Hoechst, fluorescein isothiocyanate, and cytochrome c dyes showed an increase in intensity that was dependent on the increase of liriodenine concentration, suggesting cell permeability was disrupted, allowing release of cytochrome c into the cytosol, while the mitochondrial membrane potential and cell numbers showed a reduction in fluorescence intensity. Average fluorescence intensities were observed simultaneously in CAOV-3 cells for all parameters. All data are shown as the mean ± standard deviation. Statistical significance was expressed at *P<0.05 (magnification 20×).
Mentions: The Multiparameter Cytotoxicity 3 assay (Cellomics) was carried out to investigate four parameters that are important in the apoptosis process, ie, total nuclear intensity, mitochondrial membrane potential, cell permeability, and cytochrome c release (Figure 6). The results indicated a reduction in total nuclei (Hoechst dye), which refers to the number of CAOV-3 cells present after being treated with liriodenine for 24 hours. The same situation occurred in the mitochondrial membrane potential, whereby there was a significant reduction in intensity (P<0.05) in cells treated with liriodenine 30 and 40 μM, respectively, at 24 hours. Meanwhile, cell permeability and cytochrome c were significantly increased (P<0.05) when compared with untreated CAOV-3 cells at the same concentrations.

Bottom Line: The result showed that liriodenine inhibits proliferation of CAOV-3 cells at 37.3 μM after 24 hours of exposure.Involvement of the intrinsic pathway in the mitochondria could be seen, with a significant increase in mitochondrial permeability and cytochrome c release, whereas the mitochondrial membrane potential was decreased.These findings indicate that liriodenine could be considered as a promising anticancer agent.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.

ABSTRACT
Enicosanthellum pulchrum is a tropical plant from Malaysia and belongs to the Annonaceae family. This plant is rich in isoquinoline alkaloids. In the present study, liriodenine, an isoquinoline alkaloid, was examined as a potential anticancer agent, particularly in ovarian cancer. Liriodenine was isolated by preparative high-performance liquid chromatography. Cell viability was performed to determine the cytotoxicity, whilst the detection of morphological changes was carried out by acridine orange/propidium iodide assay. Initial and late apoptosis was examined by Annexin V-fluorescein isothiocyanate and DNA laddering assays, respectively. The involvement of pathways was detected via caspase-3, caspase-8, and caspase-9 analyses. Confirmation of pathways was further performed in mitochondria using a cytotoxicity 3 assay. Apoptosis was confirmed at the protein level, including Bax, Bcl-2, and survivin, while interruption of the cell cycle was used for final validation of apoptosis. The result showed that liriodenine inhibits proliferation of CAOV-3 cells at 37.3 μM after 24 hours of exposure. Changes in cell morphology were detected by the presence of cell membrane blebbing, chromatin condensation, and formation of apoptotic bodies. Early apoptosis was observed by Annexin V-fluorescein isothiocyanate bound to the cell membrane as early as 24 hours. Liriodenine activated the intrinsic pathway by induction of caspase-3 and caspase-9. Involvement of the intrinsic pathway in the mitochondria could be seen, with a significant increase in mitochondrial permeability and cytochrome c release, whereas the mitochondrial membrane potential was decreased. DNA fragmentation occurred at 72 hours upon exposure to liriodenine. The presence of DNA fragmentation indicates the CAOV-3 cells undergo late apoptosis or final stage of apoptosis. Confirmation of apoptosis at the protein level showed overexpression of Bax and suppression of Bcl-2 and survivin. Liriodenine inhibits progression of the CAOV-3 cell cycle in S phase. These findings indicate that liriodenine could be considered as a promising anticancer agent.

No MeSH data available.


Related in: MedlinePlus