Mustard Seed (Brassica nigra) Extract Exhibits Antiproliferative Effect against Human Lung Cancer Cells through Differential Regulation of Apoptosis, Cell Cycle, Migration, and Invasion

Abstract

Lung cancer is the primary cause of cancer-related death worldwide, and development of novel lung cancer preventive and therapeutic agents are urgently needed. Brassica nigra (black mustard) seeds are commonly consumed in several Asian and African countries. Mustard seeds previously exhibited significant anticancer activities against several cancer types. In the present study, we have investigated various cellular and molecular mechanisms of anticancer effects of an ethanolic extract of B. nigra seeds against A549 and H1299 human non-small cell lung cancer cell lines. B. nigra extract showed a substantial growth-inhibitory effect as it reduced the viability and clonogenic survival of A549 and H1299 cells in a concentration-dependent manner. B. nigra extract induced cellular apoptosis in a time- and concentration-dependent fashion as evidenced from increased caspase-3 activity. Furthermore, treatment of both A549 and H1299 cells with B. nigra extract alone or in combination with camptothecin induced DNA double-strand breaks as evidenced by upregulation of γH2A histone family member X, Fanconi anemia group D2 protein, Fanconi anemia group J protein, ataxia-telangiectesia mutated and Rad3-related protein. Based on cell cycle analysis, B. nigra extract significantly arrested A549 and H1299 cells at S and G2/M phases. Additionally, B. nigra extract suppressed the migratory and invasive properties of both cell lines, downregulated the expression of matrix metalloproteinase-2 (MMP2), MMP9, and Snail and upregulated the expression of E-cadherin at mRNA and protein levels. Taken together, these findings indicate that B. nigra seed extract may have an important anticancer potential against human lung cancer which could be mediated through simultaneous and differential regulation of proliferation, apoptosis, DNA damage, cell cycle, migration, and invasion.

In Vitro Cytotoxic Activity of African Plants: A Review

“…Different assays were performed to evaluate the cytotoxicity effects of the studied African plant extracts, including a crystal violet cell antiproliferation assay [ 29 ], resazurin reduction assay [ 20 , 21 , 22 , 25 , 34 ], or tetrazolium-based colorimetric cell proliferation assays such as MTT [ 14 , 16 , 17 , 19 , 23 , 26 , 28 , 29 , 30 , 31 , 33 ], MTS [ 32 ] and WST-1 [ 13 ]. There were studies where cell numbers were determined using Hoechst 33342/Propidium Iodide (PI) staining [ 12 , 18 ], trypan blue [ 24 ], or were based on the quantification of Adenosin Triphosphate (ATP), signaling the presence of metabolically active cells [ 15 , 27 ].…”

“…Tesfaye et al (2021) [ 29 ] showed the potent activity of two plants out of 22 collected in Ethiopia against all 10 cell lines evaluated, namely A. schimperi extract (80% methanol), with IC 50 values from 1.87 ± 0.4 to 10.31 ± 3.45 µg/mL, and K. petitiana extracts which had IC 50 values from 2.09 ± 0.43 to 10.41 ± 5.59 µg/mL [ 29 ]. Moreover, B. nigra extract, collected from Egypt and evaluated in the study of Ahmed et al (2020) [ 31 ], showed IC 50 values of 32.02 and 25.38 µg/mL against the A549 and H1299 cell lines, respectively. Sawadogo et al (2020) [ 32 ] showed for the L. ukambensis crude extract (plant collected from Burkina Faso) an IC 50 value of 23.05 ± 1.56 µg/mL in the HCT-116 cell line.…”

“…The bioassay-guided fractionation of the A. boonei extract, from Ghana, performed by Spiegler et al (2021), revealed the presence of an alkaloid (15-hydroxyangustilobine A), which, at concentrations ≥ 60 µM, caused an increase in the number of cells in the G2/M phase, which was higher than cells in apoptosis at the same concentration [ 28 ]. Moreover, caspase-3 kinetic activity increased in A549 and H1299 cancer cell lines treated with B. nigra extract in a time-dependent manner, suggesting that the plant extract collected in Egypt has the ability to induce apoptosis [ 31 ]. Ahmed et al (2020) also showed that B. nigra ethanolic extract significantly delayed and arrested, at the S and G2/M transition, both A549 and H1299 cells in a concentration-dependent manner [ 31 ].…”

Anticancer effects of Poncirus fructus on hepatocellular carcinoma through regulation of apoptosis, migration, and invasion

“…Equal amounts of protein mixed with non-reducing 6X loading buffer were separated by SDS-PAGE containing 0.1% gelatin. After electrophoresis, the gels were rinsed thrice with 0.25% Triton X-100 for 15 min at room temperature and then incubated at 37°C in developing buffer (50 mM Tris-HCL pH 7.4, 5mM CaCl 2 , 200 mM NaCl) for 42 h. After incubation, the MMP-2 and MMP-9 activities were measured by Coomassie blue staining and the gel was imaged using an LAS 3000 (Fuji) ( 39 ).…”

Edible Seeds Medicinal Value, Therapeutic Applications and Functional Properties-a Review

“…B. nigra extract showed a substantial growth-inhibitory effect as it reduced the viability and clonogenic survival of A549 and H1299 cells in a concentration-dependent manner. B. nigra Antiasthmatic activity of mustard seed seed extract may be an important anticancer potential against human lung cancer [24].…”