Anti-proliferative effect and underlying mechanism of ethoxy-substituted phylloquinone (vitamin K1 derivative) from Spinacia oleracea leaf and enhancement of its extractability using radiation technology

Abstract

In our previous studies, a novel antimutagenic compound, 2-ethoxy-3-(3,7,11,15-tetramethylhexadec-2-ethyl) naphthaquinone-1,4-dione (ethoxy-substituted phylloquinone; ESP) from spinach was characterized and mechanism contributing to its antimutagenicity was deduced. In the current study, anti-proliferative activity of ESP was assessed in lung cancer (A549) cells using MTT [3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide], clonogenic assays and cell cycle analysis. ESP treatment showed selective cytotoxicity against lung cancer cells and no cytotoxicity in normal lung (WI38) cells. Cell cycle analysis revealed that ESP treatment arrests A549 cell population in G2-M phase. In-silico analysis indicated positive drug-likeness features of ESP. Molecular docking showed H-bonding and hydrophobic interactions between ESP and B-DNA dodecamer residues at minor groove. SWATH-MS (Sequential Window Acquisition of All Theoretical Mass Spectra) based proteomic analysis indicated down-regulation of proteins involved in EGFR signaling, NEDDylation and other metabolic pathways and up-regulation of tumor suppressor (STAT1 and NDRG1) proteins. Treatment of spinach powder with gamma radiation (5-20 kGy) from cobalt (Co-60) enhanced the extractability of ESP up to 4.4-fold at the highest dose of 20 kGy. Scanning electron microscopy of spinach powder displayed decrease in smoothness and compactness with increase in radiation dose attributing to its enhanced extractability. Increase in the extractability of ESP with increasing radiation doses as measured by fluorescence intensity and dry weight basis was strongly correlated. Nonetheless, radiation treatment did not affect the functionality of ESP in terms of anti-proliferative and antimutagenic activities. Current findings thus highlight broad spectrum bioactivity of ESP from spinach, its underlying mechanism and applicability of radiation technology in enhancing extractability.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-022-03264-6.

Keywords: Lung cancer cell; Minor groove binding; Molecular docking; Radiation technology; SWATH-MS.

Fig. 1

Assessment of cytotoxicity of ESP in lung cancer cells (A549) (A) and normal lung cells (WI38) (B) using MTT assay. Different letters (a–e) on the top of the bars showed significant differences among means (p ≤ 0.05)

Fig. 2

Time-dependent microscopic examination (A) and clonogenic assay of ESP against lung cancer (A549) cells (B) and normal lung (WI38) cells (C); Histogram showing effect of ESP on colony count and size of A549 cells (D), Histogram showing effect of ESP on A549 cells cell population (%) during cell cycle analysis by flow cytometry (E). Different letters (a–d) on the top of the bars showed significant differences among means (p ≤ 0.05)

Fig. 2

Time-dependent microscopic examination (A) and clonogenic assay of ESP against lung cancer (A549) cells (B) and normal lung (WI38) cells (C); Histogram showing effect of ESP on colony count and size of A549 cells (D), Histogram showing effect of ESP on A549 cells cell population (%) during cell cycle analysis by flow cytometry (E). Different letters (a–d) on the top of the bars showed significant differences among means (p ≤ 0.05)

Fig. 3

Interaction of ESP at minor groove of B-DNA (A) and possible site of interactions at minor groove (B) using Chain A—deoxyC₁GCGAATTCGCG₁₂; Chain B—deoxyC₁₃GCGAATTCGCG₂₄ ;  H-bond;   Hydrophobic interactions

Fig. 4

Functional enrichment analysis using gProfiler for the Gene Ontology (GO) and biological pathways using down-regulated (A) and up-regulated (B) proteins

Fig. 5

Model explaining possible mechanism of anti-proliferative activity of ESP based on SWATH-MS proteomic analysis. Down-regulation (≥ 20-fold) of RalA, NEDD8, DDX21 and other proteins (TFAM, SRSF3, STMN1, EIF1 & 3G, ELOC, etc.) may inhibit the cell proliferation /survival by finally affecting the replication, transcription, translation, splicing and cell progression. Upregulated (≥ fivefold) STAT1 and NDRG1 possibly inhibited cell progression and F-actin polymerization required for cell division, respectively Normal Pathway (in absence of ESP);   Inhibition (in presence of ESP)

Fig. 6

Schematic representation of extraction process with possible explanation involved in isolating ESP from spinach powder (A); Extractability of ESP from irradiated in the range of 5 to 20 kGy of spinach powder in terms of fluorescence intensity upon TLC (inset) and yield (weight; µg/g) (B); Scanning electron microscopy (SEM) of irradiated (5–20 kGy) spinach powder (C); Functionality [antimutagenicity (D) and anti-proliferative activity based on MTT assay (E)] of ESP from irradiated spinach powder. Different letters (a–d) on the top of the bars showed significant differences among means (p ≤ 0.05)

Fig. 6

Schematic representation of extraction process with possible explanation involved in isolating ESP from spinach powder (A); Extractability of ESP from irradiated in the range of 5 to 20 kGy of spinach powder in terms of fluorescence intensity upon TLC (inset) and yield (weight; µg/g) (B); Scanning electron microscopy (SEM) of irradiated (5–20 kGy) spinach powder (C); Functionality [antimutagenicity (D) and anti-proliferative activity based on MTT assay (E)] of ESP from irradiated spinach powder. Different letters (a–d) on the top of the bars showed significant differences among means (p ≤ 0.05)