Securinine from Phyllanthus glaucus Induces Cell Cycle Arrest and Apoptosis in Human Cervical Cancer HeLa Cells

Abstract

BackgroundThe Securinega-type alkaloids occur in plants belonging to Euphorbiaceae family. One of the most widely distributed alkaloid of this group is securinine, which was identified next to allosecurinine in Phyllanthus glaucus (leafflower). Recently, some Securinega-type alkaloids have paid attention to its antiproliferative potency towards different cancer cells. However, the cytotoxic properties of allosecurinine have not yet been evaluated.MethodsThe cytotoxicity of the extract, alkaloid fraction obtained from P. glaucus, isolated securinine and allosecurinine against HeLa cells was evaluated by real-time xCELLigence system and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by annexin V and 7-amino-actinomycin (7-AAD) staining and confirmed with fluorescent Hoechst 33342 dye. The assessment of mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, the level of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), caspase-3/7 activity and cell cycle analysis were measured by flow cytometry. The enzymatic activity of caspase-9 was assessed by a luminometric assay. The expression of apoptosis associated genes was analyzed by real-time PCR.ResultsThe experimental data revealed that securinine and the alkaloid fraction were significantly potent on HeLa cells growth inhibition with IC50 values of 7.02 ± 0.52 μg/ml (32.3 μM) and 25.46 ± 1.79 μg/ml, respectively. The activity of allosecurinine and Phyllanthus extract were much lower. Furthermore, our study showed that the most active securinine induced apoptosis in a dose-dependent manner in the tested cells, increased the percentage of ROS positive cells and depolarized cells as well as stimulated the activity of ERK1/2, caspase-9 and -3/7. Securinine also induced cell cycle arrest in S phase. Real-time PCR analysis showed high expression of TNFRSF genes in the cells stimulated with securinine.ConclusionsSecurinine induces apoptosis and activates cell cycle checkpoints in HeLa cells which is associated with oxidative stress. The results indicate that the mitochondrial pathway is involved in the programmed cell death.

Orostachys japonicus induce p53‐dependent cell cycle arrest through the MAPK signaling pathway in OVCAR‐3 human ovarian cancer cells

“…The cell cycle process is basically carried out in the order of the G1, S, and G2/M phases. When apoptosis is stimulated, the cell cycle process can be stopped at several stages(Stefanowicz-Hajduk, Sparzak-Stefanowska, Krauze-Baranowska, & Ochocka, 2016). Based on this, the effect of OJE on the cell cycle phase of OVCAR-3 cells was determined by flow cytometry.…”

Influence of plant growth regulators on the shoot culture of Phyllanthus glaucus and accumulation of indolizidine alkaloids with evaluation of antimicrobial activity

“…Thermolabile plant growth regulators [PGRs; indole-3-acetic acid (IAA), and 2-isopentenyladenine (2iP)] were added postautoclaving via sterile filtration (0.2 µm filters, FP 3070, 2 CA-S, Schleicher and Schuell, Keene, NH, USA). The other aspects of preparation of culture media and growth conditions were described previously (Stefanowicz-Hajduk et al 2016).…”

“…The origin of the plant material and explants preparation has been described in earlier work (Sparzak et al 2015;Stefanowicz-Hajduk et al 2016).…”

“…Preparation of dry extract was performed according to previously established method (Stefanowicz-Hajduk et al 2016).…”

“…Recently, the cytotoxic activity of securinine was investigated against several cancer cell lines-breast (MCF-7), colon , and leukemia (HL-60) (Dong et al 1999;Li et al 2014;Rana et al 2010;Xia et al 2011). The study showed that securinine possesses significantly higher activity to the HeLa cells than allosecurinine with IC 50 values of 7.02 ± 0.52 µg/mL (32.3 µM) and 52.88 ± 4.62 µg/mL (243.4 µM), respectively (Stefanowicz-Hajduk et al 2016).…”