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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

Fluorescent micrographs and quantitative analysis of CAOV-3 cells double-stained with acridine orange and propidium iodide. Cells were treated with liriodenine in a time-dependent manner.Notes: (A) Untreated cells without any morphological changes. (B) Cells treated for 24 hours show early apoptosis by the presence of bright green color (acridine orange). (C) Cells treated for 48 hours show more membrane cell blebbing and chromatin condensation. (D) Cells treated for 72 hours show more orange-colored staining, indicating late apoptosis. (A–D) Magnification 40×. (E) Histogram of three cell phases. *P<0.05 indicates a significant difference from control.Abbreviations: VI, viable cells; BL, blebbing of cell membrane; CC, chromatin condensation; EA, early apoptosis; LA, late apoptosis; AB, apoptotic bodies; VS, production of vesicles.
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f3-dddt-9-1437: Fluorescent micrographs and quantitative analysis of CAOV-3 cells double-stained with acridine orange and propidium iodide. Cells were treated with liriodenine in a time-dependent manner.Notes: (A) Untreated cells without any morphological changes. (B) Cells treated for 24 hours show early apoptosis by the presence of bright green color (acridine orange). (C) Cells treated for 48 hours show more membrane cell blebbing and chromatin condensation. (D) Cells treated for 72 hours show more orange-colored staining, indicating late apoptosis. (A–D) Magnification 40×. (E) Histogram of three cell phases. *P<0.05 indicates a significant difference from control.Abbreviations: VI, viable cells; BL, blebbing of cell membrane; CC, chromatin condensation; EA, early apoptosis; LA, late apoptosis; AB, apoptotic bodies; VS, production of vesicles.

Mentions: Morphological changes in CAOV-3 cells were observed under the fluorescence microscope after exposure to liriodenine. These changes could be seen at 24, 48, and 72 hours (Figure 3). In comparison with untreated CAOV-3 cells, the treated cells showed an intact green nuclear structure with a round shape and no disruption. After 24 hours of treatment with liriodenine, the morphology of CAOV-3 cells showed cell membrane blebbing and fragmented DNA with bright green fluorescence. The changes could be clearly observed after 48 and 72 hours of treatment, with clear growth inhibition, increased cell membrane blebbing, presence of more apoptotic bodies, and also the appearance of a reddish-orange color due to PI being bound to denatured DNA cells, indicating dead cells. Furthermore, the numbers of CAOV-3 cells that underwent early and late apoptosis increased in a time-dependent manner.


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)

Fluorescent micrographs and quantitative analysis of CAOV-3 cells double-stained with acridine orange and propidium iodide. Cells were treated with liriodenine in a time-dependent manner.Notes: (A) Untreated cells without any morphological changes. (B) Cells treated for 24 hours show early apoptosis by the presence of bright green color (acridine orange). (C) Cells treated for 48 hours show more membrane cell blebbing and chromatin condensation. (D) Cells treated for 72 hours show more orange-colored staining, indicating late apoptosis. (A–D) Magnification 40×. (E) Histogram of three cell phases. *P<0.05 indicates a significant difference from control.Abbreviations: VI, viable cells; BL, blebbing of cell membrane; CC, chromatin condensation; EA, early apoptosis; LA, late apoptosis; AB, apoptotic bodies; VS, production of vesicles.
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Related In: Results  -  Collection

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f3-dddt-9-1437: Fluorescent micrographs and quantitative analysis of CAOV-3 cells double-stained with acridine orange and propidium iodide. Cells were treated with liriodenine in a time-dependent manner.Notes: (A) Untreated cells without any morphological changes. (B) Cells treated for 24 hours show early apoptosis by the presence of bright green color (acridine orange). (C) Cells treated for 48 hours show more membrane cell blebbing and chromatin condensation. (D) Cells treated for 72 hours show more orange-colored staining, indicating late apoptosis. (A–D) Magnification 40×. (E) Histogram of three cell phases. *P<0.05 indicates a significant difference from control.Abbreviations: VI, viable cells; BL, blebbing of cell membrane; CC, chromatin condensation; EA, early apoptosis; LA, late apoptosis; AB, apoptotic bodies; VS, production of vesicles.
Mentions: Morphological changes in CAOV-3 cells were observed under the fluorescence microscope after exposure to liriodenine. These changes could be seen at 24, 48, and 72 hours (Figure 3). In comparison with untreated CAOV-3 cells, the treated cells showed an intact green nuclear structure with a round shape and no disruption. After 24 hours of treatment with liriodenine, the morphology of CAOV-3 cells showed cell membrane blebbing and fragmented DNA with bright green fluorescence. The changes could be clearly observed after 48 and 72 hours of treatment, with clear growth inhibition, increased cell membrane blebbing, presence of more apoptotic bodies, and also the appearance of a reddish-orange color due to PI being bound to denatured DNA cells, indicating dead cells. Furthermore, the numbers of CAOV-3 cells that underwent early and late apoptosis increased in a time-dependent manner.

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