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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.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.

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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In vitro activity of naringenin against Human skin carcinoma A431 and normal HaCat cells.(A) Morphological view of live and dead cells of A431 cell line treated with 50 µM to 750 µM concentration of naringenin (B) The percent cell viability of A431 cells measured by a MTT assay at 24 h as described in the experimental section. (C) Morphological analysis of normal HaCat cell line treated with 50 µM to 750 µM concentration of naringenin. Photomicrographs were taken by inverted phase contrast microscope. (D) The percent cell viability of HaCat cell measured by a MTT assay at 24 h. 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-g001: In vitro activity of naringenin against Human skin carcinoma A431 and normal HaCat cells.(A) Morphological view of live and dead cells of A431 cell line treated with 50 µM to 750 µM concentration of naringenin (B) The percent cell viability of A431 cells measured by a MTT assay at 24 h as described in the experimental section. (C) Morphological analysis of normal HaCat cell line treated with 50 µM to 750 µM concentration of naringenin. Photomicrographs were taken by inverted phase contrast microscope. (D) The percent cell viability of HaCat cell measured by a MTT assay at 24 h. 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: To find out the experimental doses of naringenin, cytotoxic tests were performed on normal skin cells HaCaT using MTT assay and cell morphology assessment. Morphological study suggests that higher concentration 750 µM of naringenin changes cell morphology and induces cell death. However, doses 50 µM, 100 µM, 200 µM, 300 µM, 400 µM and 500 µM of naringenin did not affect significantly on cell morphology as revealed in Fig. 1C. Cell viability data showed that percent cell viability in normal cells was 91.97% and 85.40% at 400 µM and 500 µM concentrations of naringenin respectively i.e. cell death was statistically insignificant, however, significant cell death was observed at 750 µM of naringenin (Fig. 1D).


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)

In vitro activity of naringenin against Human skin carcinoma A431 and normal HaCat cells.(A) Morphological view of live and dead cells of A431 cell line treated with 50 µM to 750 µM concentration of naringenin (B) The percent cell viability of A431 cells measured by a MTT assay at 24 h as described in the experimental section. (C) Morphological analysis of normal HaCat cell line treated with 50 µM to 750 µM concentration of naringenin. Photomicrographs were taken by inverted phase contrast microscope. (D) The percent cell viability of HaCat cell measured by a MTT assay at 24 h. 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-g001: In vitro activity of naringenin against Human skin carcinoma A431 and normal HaCat cells.(A) Morphological view of live and dead cells of A431 cell line treated with 50 µM to 750 µM concentration of naringenin (B) The percent cell viability of A431 cells measured by a MTT assay at 24 h as described in the experimental section. (C) Morphological analysis of normal HaCat cell line treated with 50 µM to 750 µM concentration of naringenin. Photomicrographs were taken by inverted phase contrast microscope. (D) The percent cell viability of HaCat cell measured by a MTT assay at 24 h. 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: To find out the experimental doses of naringenin, cytotoxic tests were performed on normal skin cells HaCaT using MTT assay and cell morphology assessment. Morphological study suggests that higher concentration 750 µM of naringenin changes cell morphology and induces cell death. However, doses 50 µM, 100 µM, 200 µM, 300 µM, 400 µM and 500 µM of naringenin did not affect significantly on cell morphology as revealed in Fig. 1C. Cell viability data showed that percent cell viability in normal cells was 91.97% and 85.40% at 400 µM and 500 µM concentrations of naringenin respectively i.e. cell death was statistically insignificant, however, significant cell death was observed at 750 µM of naringenin (Fig. 1D).

Bottom Line: The search for antiproliferative agents that reduce skin carcinoma is a task of great importance.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.

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