Magnoflorine-Isolation and the Anticancer Potential against NCI-H1299 Lung, MDA-MB-468 Breast, T98G Glioma, and TE671 Rhabdomyosarcoma Cancer Cells

Abstract

Magnoflorine (MGN) is a quaternary aporphine alkaloid that exhibits numerous therapeutic properties, including neuropsychopharmacological, anti-anxiety, immunomodulatory, anti-inflammatory, antioxidant, or antifungal activities. The aim of the present study was an investigation of the influence of MGN on viability, proliferation, induction of apoptosis, and cell cycle arrest in NCI-H1299 lung, MDA-MB-468 breast, T98G glioma, and TE671 rhabdomyosarcoma cancer cells. MGN was isolated from the roots of Berberis cretica L. by counter-current partition chromatography (CPC). Cell viability and proliferation assessments were performed by means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and 5-bromo-2'-deoxyuridine (BrDU) assays, respectively. The induction of apoptosis and cell cycle progression was measured using fluorescence-activated cell sorting analysis. MGN in high doses inhibits proliferation, induces apoptosis, and inhibits cell cycle in S/G2 phases in a dose-dependent manner. MGN seems to be a promising anti-cancer compound in therapy of some types of lung, breast, glioma, and rhabdomyosarcoma cancers, for which current standard therapies are limited or have severe strong side effects.

Keywords: Berberidaceae; Berberis cretica; CPC; HPLC-MS; anti-cancer activity; counter-current chromatography; isoquinoline alkaloids; magnoflorine; natural products.

Figure 1

The total ion chromatogram of the methanolic extract from Berberis cretica roots.

Figure 2

The part of counter-current partition chromatography (CPC)-UV chromatogram (at 290 nm) of the Berberis cretica root extract that show the elution of magnoflorine (MGN) (A) and the total ion chromatogram (TIC) of the isolated MGN (B).

Figure 3

Effect of magnoflorine (MGN) on viability of human lung cancer (A549 (A,B) and H1299 (A) [1]), breast cancer (MCF7 (A,D) and MDA-MB-468 (A) [1]), cervix cancer (HeLa) (A,C), glioma (T98G (A) [1]), rhabdomyosarcoma (TE671 (A) [1]), and human skin fibroblast (HSF (A,E)) cells. The cancer cells were exposed to either culture medium alone (control) or magnoflorine (0.01–2 mg/mL) for 96 h. Normalized cell viability measured by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay is presented as mean ± SD at each concentration. The differences between groups were evaluated using the one-way ANOVA; Tukey’s post-hoc test. *** p < 0.001, ** p < 0.01, * p < 0.05; n = 18 per concentration from three independent experiments.

Figure 4

Effect of magnoflorine (MGN) on the proliferation of T98G glioma (A,E), MDA-MB-468 breast (B,E), NCIH1299 lung (C,E), and TE671 rhabdomyosarcoma (D,E) cancer cells in 5-bromo-2ʹ-deoxyuridine (BrdU) assay. Cancer cells were incubated for 48 h alone (control) or in the presence of magnoflorine (0.01–1 mg/mL). The differences between groups were evaluated using the one-way ANOVA; Tukey’s post-hoc test. *** p < 0.001. Results from three independent experiments were presented as mean ± SD of the mean.

Figure 5

Effects of magnoflorine (MGN) on caspase-3 activation in T98G glioma (A), MDA-MB-468 breast (B), NCIH1299 lung (C), and TE671 rhabdomyosarcoma (D) cancer cells. Cancer cell lines were cultivated for 48 h with different doses of MGN (5–10 mg/mL) and analyzed by flow cytometry. The values present the percentage of the cells with active caspase-3. The results are depicted as means ± SD, n = 9 from three separate experiments. Statistical analysis was performed using one-way ANOVA test. *** p < 0.001.

Figure 6

Representative dot plots of the flow cytometry analysis of T98G glioma cells after 48 h treatment with medium (ctr) (A) or magnoflorine (5 mg/mL) (B), 7.5 mg/mL (C), and 10 mg/mL (D); n = 9 per concentration from three independent experiments. R2-cells with active caspase-3 (apoptotic cells).

Figure 7

Effects of magnoflorine (MGN) on the cell cycle progression in NCIH1299 lung (A), MDA-MB-468 breast (B), T98G glioma (C), and TE671 rhabdomyosarcoma (D) cancer cells. Cancer cell lines were cultivated for 48 h with different doses of magnoflorine (5–10 mg/mL) and analyzed by flow cytometry. The results are depicted as means ± SD, n = 9 from three separate experiments.