Apoptosis is a highly regulated, organized, and programmed death process controlling the development and homeostasis of cells in multicellular organisms . manifestation of cyclinD1, cyclinE1, and cyclin-dependent kinase 2 in the protein level. The effect of hesperidin was also verified within the human being colon cancer cell HT-29 cells. Conclusion We concluded that hesperidin inhibited HeLa cell proliferation through apoptosis including endoplasmic reticulum stress pathways and cell cycle arrest. values less than 0.05 were considered significant. Results HES-induced morphological changes and anti-proliferation effect in HeLa cells and HT-29 cells HeLa cells and HT-29 cells were incubated with HES (0, 20, 40, 60, 80, and 100?M) for 48?h. The morphology of the cells was examined using a phase contrast microscope. In the presence of HES, HeLa cells showed round morphology with a small amount of shrinkage and nuclear condensation, and a proportion of the cells showed swelling, cell membrane lysis, and disintegration of organelles, suggesting HES-induced toxicity to HeLa cells (Fig.?1a and c). Open in a separate windows Fig. 1 Hesperidin (HES)-induced morphological switch and anti-proliferation in HeLa cells and HT-29 cells. a and c The morphology of the HeLa cells and HT-29 cellswas examined using a phase contrast Acetohexamide microscope after treatment with HES. After treatment with HES (0, 20, 40, 60, 80, and 100?M) for 48?h, Cells showed several morphological changes. <0.05 versus control group (0?M) (two-way ANOVA followed by Tukeys post hoc test) Cell viability was TNFSF13B evaluated from the MTT assay at 24, 48, and 72?h and results were reported while family member cell viability (%). All data were normalized to the control group (100?%). Treatment with HES significantly reduced cell viability compared to the control group (Fig.?1b and d) and the effect of HES about cell viability was concentration-and time-dependent. Cells incubated with 100?M HES for 72?h showed the maximum anti-proliferative effect, with cell viability decreased to 12?% of the control cells. This result suggests that HES inhibits proliferation of HeLa cells inside a concentration- and time-dependent manner. HES-induced apoptosis in HeLa cells and HT-29 cells HeLa cells and HT-29 cells were treated with HES (0, 40, 80, and 160?M) for 48 hand apoptosis was assessed with Hoechst 33342 apoptosis detection kit. Representative images of Hoechst 33342 staining are demonstrated in Fig.?2a and c. HES-treated cells exhibited standard morphological changes indicating apoptosis. The nuclei with condensed chromatin showed more fluorescence than the nuclei in normal cells. Apoptotic HeLa cells also displayed round and Acetohexamide shrunken cell body (white arrows in Fig.?2a and c). The number of apoptotic HeLa cells Acetohexamide improved as the concentration of HES improved (Fig.?2b and d), suggesting that HES-induced apoptosis of HeLa cells might contribute to reduced cell viability. Open in a separate windows Fig. 2 HeLa cell and HT-29 cell apoptosis after treatment with hesperidin (HES) observed using Hoechst 33342 staining. a and c HeLa cells and HT-29 cells were treated with HES (0, 40, 80, and 160?M) for 48?h. Apoptotic cells (<0.05 versus control group (0?M) (one-way ANOVA followed by Tukeys post hoc test) HES-induced DNA fragmentation in HeLa cells DNA fragmentation is considered another hallmark of apoptosis. HeLa cells were treated with HES (0, 40, 80, and 160?M) for 48?h and DNA fragmentation was detected using the DNA laddering fragmentation assay. The cleaved DNA fragments in apoptotic HeLa cells were separated by agarose gel electrophoresis (Fig.?3). Staining of the gel with ethidium bromide exposed typical laddering pattern of multimers of 500C1000 bases. Treatment with 80 and 160?M HES markedly increased DNA fragmentation in HeLa cells. HES induced DNA fragmentation inside a concentration-dependent manner. Open in a separate windows Fig. 3 DNA fragmentation as an apoptotic effect of hesperidin (HES) in HeLa cells. HeLa cells were treated with HES (0, 40, 80, and 160?M) for 48?h and DNA fragmentation was determined using DNA gel electrophoresis HES-induced increase in ROS and cytoplasmic Ca2+ levels and decrease in m in HeLa cells To evaluate HES-induced oxidative stress in HeLa cells, the level of ROS was detected by circulation cytometry after cells were treated with HES (0, 40, 80, and 160?M) for 48?h. The level of ROS was improved in the HES-treated organizations inside a concentration-dependent manner. ROS production was maximal after treatment with 160?M HES (Fig.?4a). Open in a separate windows Fig. 4 Hesperidin (HES).
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