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Induction of apoptosis and antiproliferative activity of naringenin in human epidermoid carcinoma cell through ROS generation and cell cycle arrest.

Ahamad MS, Siddiqui S, Jafri A, Ahmad S, Afzal M, Arshad M - PLoS ONE (2014)

Bottom Line: The search for antiproliferative agents that reduce skin carcinoma is a task of great importance.The intracellular ROS generation assay showed statistically significant (p<0.001) dose-related increment in ROS production for naringenin.Cell cycle study showed that naringenin induced cell cycle arrest in G0/G1 phase of cell cycle and caspase-3 analysis revealed a dose dependent increment in caspase-3 activity which led to cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Shibli National (PG) College, Azamgarh, Uttar Pradesh, India.

ABSTRACT
A natural predominant flavanone naringenin, especially abundant in citrus fruits, has a wide range of pharmacological activities. The search for antiproliferative agents that reduce skin carcinoma is a task of great importance. The objective of this study was to analyze the anti-proliferative and apoptotic mechanism of naringenin using MTT assay, DNA fragmentation, nuclear condensation, change in mitochondrial membrane potential, cell cycle kinetics and caspase-3 as biomarkers and to investigate the ability to induce reactive oxygen species (ROS) initiating apoptotic cascade in human epidermoid carcinoma A431 cells. Results showed that naringenin exposure significantly reduced the cell viability of A431 cells (p<0.01) with a concomitant increase in nuclear condensation and DNA fragmentation in a dose dependent manner. The intracellular ROS generation assay showed statistically significant (p<0.001) dose-related increment in ROS production for naringenin. It also caused naringenin-mediated epidermoid carcinoma apoptosis by inducing mitochondrial depolarization. Cell cycle study showed that naringenin induced cell cycle arrest in G0/G1 phase of cell cycle and caspase-3 analysis revealed a dose dependent increment in caspase-3 activity which led to cell apoptosis. This study confirms the efficacy of naringenin that lead to cell death in epidermoid carcinoma cells via inducing ROS generation, mitochondrial depolarization, nuclear condensation, DNA fragmentation, cell cycle arrest in G0/G1 phase and caspase-3 activation.

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Photomicrographs showing intracellular ROS generation in A431 cells induced by naringenin and stained with DCFH-DA.(A) Photomicrographs showing intracellular ROS generation induced by 100 µM, 300 µM and 500 µM of naringenin after 12 h incubation. Photomicrographs were taken by florescence phase contrast microscope (B) Values are expressed as the percentage of fluorescence intensity relative to the control. Values are expressed as means ± SD of at least three independent experiments, **p<0.01 and **p<0.001 as compared with their respective control.
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pone-0110003-g003: Photomicrographs showing intracellular ROS generation in A431 cells induced by naringenin and stained with DCFH-DA.(A) Photomicrographs showing intracellular ROS generation induced by 100 µM, 300 µM and 500 µM of naringenin after 12 h incubation. Photomicrographs were taken by florescence phase contrast microscope (B) Values are expressed as the percentage of fluorescence intensity relative to the control. Values are expressed as means ± SD of at least three independent experiments, **p<0.01 and **p<0.001 as compared with their respective control.

Mentions: ROS generation was assessed at 12 h after naringenin treatment. Florescent micrograph of stained A431 cells (Fig. 3) depicts the effect of naringenin-induced intracellular ROS generation. The photomicrograph suggests that A431 cells treated with naringenin elevates ROS intensity in a dose dependent manner as compared to control (Fig. 3A). Maximum ROS generation was observed in the cells treated at 500 µM of naringenin. The quantitative measurement of ROS intensity showed that 100 µM of naringenin elevates significant ROS production, approximately 7.43% (**p<0.01) as compared to control. However, ROS production was sustained increasingly to approximately 17.46% and 51.52% (**p<0.001) at the concentrations 300 µM and 500 µM of naringenin, respectively (Fig. 3B). Increased ROS generation is involved in the induction of apoptosis through various pathways.


Induction of apoptosis and antiproliferative activity of naringenin in human epidermoid carcinoma cell through ROS generation and cell cycle arrest.

Ahamad MS, Siddiqui S, Jafri A, Ahmad S, Afzal M, Arshad M - PLoS ONE (2014)

Photomicrographs showing intracellular ROS generation in A431 cells induced by naringenin and stained with DCFH-DA.(A) Photomicrographs showing intracellular ROS generation induced by 100 µM, 300 µM and 500 µM of naringenin after 12 h incubation. Photomicrographs were taken by florescence phase contrast microscope (B) Values are expressed as the percentage of fluorescence intensity relative to the control. Values are expressed as means ± SD of at least three independent experiments, **p<0.01 and **p<0.001 as compared with their respective control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199682&req=5

pone-0110003-g003: Photomicrographs showing intracellular ROS generation in A431 cells induced by naringenin and stained with DCFH-DA.(A) Photomicrographs showing intracellular ROS generation induced by 100 µM, 300 µM and 500 µM of naringenin after 12 h incubation. Photomicrographs were taken by florescence phase contrast microscope (B) Values are expressed as the percentage of fluorescence intensity relative to the control. Values are expressed as means ± SD of at least three independent experiments, **p<0.01 and **p<0.001 as compared with their respective control.
Mentions: ROS generation was assessed at 12 h after naringenin treatment. Florescent micrograph of stained A431 cells (Fig. 3) depicts the effect of naringenin-induced intracellular ROS generation. The photomicrograph suggests that A431 cells treated with naringenin elevates ROS intensity in a dose dependent manner as compared to control (Fig. 3A). Maximum ROS generation was observed in the cells treated at 500 µM of naringenin. The quantitative measurement of ROS intensity showed that 100 µM of naringenin elevates significant ROS production, approximately 7.43% (**p<0.01) as compared to control. However, ROS production was sustained increasingly to approximately 17.46% and 51.52% (**p<0.001) at the concentrations 300 µM and 500 µM of naringenin, respectively (Fig. 3B). Increased ROS generation is involved in the induction of apoptosis through various pathways.

Bottom Line: The search for antiproliferative agents that reduce skin carcinoma is a task of great importance.The intracellular ROS generation assay showed statistically significant (p<0.001) dose-related increment in ROS production for naringenin.Cell cycle study showed that naringenin induced cell cycle arrest in G0/G1 phase of cell cycle and caspase-3 analysis revealed a dose dependent increment in caspase-3 activity which led to cell apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Shibli National (PG) College, Azamgarh, Uttar Pradesh, India.

ABSTRACT
A natural predominant flavanone naringenin, especially abundant in citrus fruits, has a wide range of pharmacological activities. The search for antiproliferative agents that reduce skin carcinoma is a task of great importance. The objective of this study was to analyze the anti-proliferative and apoptotic mechanism of naringenin using MTT assay, DNA fragmentation, nuclear condensation, change in mitochondrial membrane potential, cell cycle kinetics and caspase-3 as biomarkers and to investigate the ability to induce reactive oxygen species (ROS) initiating apoptotic cascade in human epidermoid carcinoma A431 cells. Results showed that naringenin exposure significantly reduced the cell viability of A431 cells (p<0.01) with a concomitant increase in nuclear condensation and DNA fragmentation in a dose dependent manner. The intracellular ROS generation assay showed statistically significant (p<0.001) dose-related increment in ROS production for naringenin. It also caused naringenin-mediated epidermoid carcinoma apoptosis by inducing mitochondrial depolarization. Cell cycle study showed that naringenin induced cell cycle arrest in G0/G1 phase of cell cycle and caspase-3 analysis revealed a dose dependent increment in caspase-3 activity which led to cell apoptosis. This study confirms the efficacy of naringenin that lead to cell death in epidermoid carcinoma cells via inducing ROS generation, mitochondrial depolarization, nuclear condensation, DNA fragmentation, cell cycle arrest in G0/G1 phase and caspase-3 activation.

Show MeSH
Related in: MedlinePlus