Brain Tumor Res Treat.  2019 Apr;7(1):25-32. 10.14791/btrt.2019.7.e21.

Bacoside A Induced Sub-G0 Arrest and Early Apoptosis in Human Glioblastoma Cell Line U-87 MG through Notch Signaling Pathway

• Aithal MG
• Rajeswari

• KMID: 2444792
• DOI: http://doi.org/10.14791/btrt.2019.7.e21

Abstract

BACKGROUND
Glioblastoma multiforme (GBM) is a highly malignant brain tumor with a worst prognosis of less than one year despite advance treatment facilities. Among various signaling pathway genes displaying genetic modifications, aberrant expression of Notch pathway genes is frequent in GBM offering novel therapeutic targets. Herbal extracts having anticancer properties are used in adjuvant therapy that is safe and affordable as compared to chemotherapeutics. Bacopa monnieri has been used for the development of brain cells because of its neuroprotective properties. Its anticancer properties have shown to be promising in cancer treatment.
METHODS
The anticancer properties of Bacoside A, an active and abundant component of Bacopa monnieri was assessed on U-87 MG cell line and its effects on expression of Notch pathway genes were studied. Cell cycle arrest and apoptosis were studied using flow cytometry. Expression of Notch pathway genes comprising of Notch receptors (notch1, notch2, notch3 and notch4), ligands (jagged1 and jagged2), a component of gamma-secretase complex (APH1A) and downstream target (HES1) were evaluated by quantitative real-time PCR.
RESULTS
Bacoside A exhibited considerable cytotoxicity on U-87 MG cells inducing cell cycle arrest and apoptosis. Cell cycle analysis revealed a significant arrest of 39.21% cells in sub-G0 phase at 80 µg/mL concentration, increasing to 53.21% at a higher concentration of 100 µg/mL. The fraction of early apoptotic cells in control was low (3.48%) that increased substantially to 31.36% and 41.11% after 80 µg/mL and 100 µg/mL of Bacoside A treatment respectively. Additionally, the expression of notch1 gene decreased after exposure to Bacoside A with a fold change of 0.05, whereas HES1 gene expression was increased by 25 fold.
CONCLUSION
These data indicate that Bacoside A has a possible anticancer activity that could be inducing cell cycle arrest and apoptosis through Notch pathway in GBM in vitro.

Keyword

Glioblastoma; Notch; Bacoside A; Cell cycle; Apoptosis; Gene expression

MeSH Terms

Amyloid Precursor Protein Secretases
Apoptosis*
Bacopa
Brain
Brain Neoplasms
Cell Cycle
Cell Cycle Checkpoints
Cell Line*
Flow Cytometry
Gene Expression
Glioblastoma*
Humans*
In Vitro Techniques
Ligands
Prognosis
Real-Time Polymerase Chain Reaction
Receptors, Notch
Amyloid Precursor Protein Secretases
Ligands
Receptors, Notch

Figure

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  Fig. 1 Effect of Bacoside A on human glioblastoma multiforme cell line U-87 MG as determined by MTT assay.

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  Fig. 2 Cell cycle profiles of untreated (A) and treated U-87 MG cells with 80 µg/mL (B) and 100 µg/mL (C) of Bacoside A respectively for 24 h as determined by flow cytometry.

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  Fig. 3 Bar graph obtained from cell cycle analysis of control, 80 µg/mL and 100 µg/mL of Bacoside A respectively showing the percentage of cells in different phases of cell cycle (G2/M, S, G0/G1 and Sub-G0).

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  Fig. 4 Apoptosis in untreated (A) and treated U-87 MG cells with 80 µg/mL (B) and 100 µg/mL (C) of Bacoside A respectively for 24 h as determined by flow cytometry.

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  Fig. 5 U-87 MG cells treated with Bacoside A stained with Annexin V-FITC/PI displaying early apoptotic cells (A), late apoptotic/necrotic cells (B) and dead cells (C) as observed under a fluorescent microscope. Scale bar, 50 µm.

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  Fig. 6 Bacoside A induced DNA fragmentation in U-87 MG cells analysed by agarose gel electrophoresis.

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  Fig. 7 Scatter plots of differentially expressed Notch pathway genes in Bacoside A treated U-87 MG cells (open circles) compared to untreated cells (filled circles) at 24 h incubation in terms of fold change derived from real-time PCR analysis.

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