Biotechnology Production of Cell Biomass from the Endangered Kickxia elatine (L.) Dumort: Its Untargeted Metabolomic Analysis and Cytotoxic Potential Against Melanoma Cells

Abstract

Background: Melanoma is a malignant tumor of melanocytes with an increasing incidence worldwide. Plant-based products are rich in bioactive compounds, offering low toxicity and accessible alternatives for melanoma treatment. A biotechnological approach to obtaining plant-derived produce ensures continuous and high-yield production of medicinally valuable biomass. Objectives: This study aimed to induce and optimize the growth of homogenous callus cultures of Kickxia elatine (L.) Dumort., consequently established a cell suspension culture with a high biomass growth rate, analyzed the phytochemical compositions, and assessed the cytotoxic activity against melanoma cells. Methods/Results: Callus cultures were induced under controlled in vitro conditions on Murashige and Skoog (MS) media supplemented with 2.0 mg L^-1 Dicamba and 2.0 mg L^-1 2,4-Dichlorophenoxyacetic acid. The selected callus lines exhibited a high growth index (351.71% ± 27.77) and showed a homogeneous morphology, beige colour, and had friable and watery characteristics. A combination of auxin and cytokinin was found to enhance biomass production significantly. Phytochemical investigations putatively annotated major compounds, including benzoic acid derivatives, phenolic glycosides, phenylpropanoic acids, hydroxycinnamic acid derivatives, and tyrosol derivatives. Methanolic extract (KE-Ex) and 40% methanolic fraction (KE-40Fr) were prepared and tested for cytotoxicity against human fibroblast (MRC-5) and melanoma (MeWo) cell lines using direct cell counting and MTT assay. The crude extract exhibited the strongest cytotoxicity effect on MeWo cells, with IC₅₀ values of 125 ± 8 µg mL^-1 after 48 h and 117 ± 7 µg mL^-1 after 72 h of treatment. Conclusions: The extract demonstrated a time- and dose-dependent cytotoxic effect, making it a potential candidate for melanoma treatment.

Keywords: callus; cell suspension culture; cytotoxic activity; melanoma cell; nuclear DNA content; phytochemical and metabolomic analyses.

Figure 1

Kickxia elatine (L.) Dumort callus induction from root (A), stem (B), leaf (C), and petiole (D) explants. Proliferation of root-derived K. elatine callus on MS media with 2.0 mg L⁻¹ Dic + 2.0 mg L⁻¹ 2,4-D (E) and the leaf-derived callus on MS supplemented with 2.0 mg L⁻¹ Dic + 0.5 mg L⁻¹ 2,4-D (F).

Figure 2

Cell suspension culture of Kickxia elatine (L.) Dumort (passage 6), established from root-derived callus, grown in MS 2.0 mg L⁻¹ Dic and 2.0 mg L⁻¹ 2,4-D and agitated at 110 RPM in a controlled room with a 16:8-hour photoperiod at 20 ± 2 °C (A). The microscopic view of K. elatine cell culture (day 18) shows clusters of small and round cells on observation using 50× (B) and 400× (C) magnification.

Figure 3

Time course of fresh (right y-axis) and dry weight (left y-axis) biomass accumulation of Kickxia elatine (L.) Dumort cell suspension culture system in 30 mL Murashige and Skoog liquid media supplemented with 2.0 mg L⁻¹ Dic + 2.0 mg L⁻¹ 2,4-D and 3% sucrose (D). The mean of fresh (black line) and dry weight (orange line) was obtained from three biological replications per observation time.

Figure 4

Histograms of nuclear DNA content obtained using flow cytometric analysis of the PI-stained nuclei isolated simultaneously from the leaves of Solanum lycopersicum L. (internal standard) and Kickxia elatine (L.) Dumort: (A) seeds, (B) in vitro-derived shoot passage 5, and (C) cell suspension culture passage 3.

Figure 5

Base peak chromatogram of Kickxia elatine (L.) Dumort calluses derived from leaf (green), stem (orange), root (blue) explants, and cell suspension culture (red) from UPLC-HRMS/MS in negative ion mode.

Figure 6

PLS-DA score plot was derived from UPLC-HRMS/MS data of secondary metabolites extracted from Kickxia elatine (L.) Dumort callus initiated from leaf (red), root (green), and stem (blue), and cell suspension culture (light blue).

Figure 7

The viability assessment of human fibroblast MRC-5 and human melanoma MeWo cells after treatment with methanolic extracts (KE-Ex), 40% MeOH fraction (KE-Fr 40%), and acteoside (KE-Ref) from Kickxia elatine (L.) Dumort callus biomass for 24, 48, and 72 h presented as a line graph. The cell viability is expressed as a percentage of the nontreated control cells. The mean of three experiments ± SD is shown. Symbols of respective levels of significance (*,•, for p < 0.05; (**, ••, ◊◊) for p < 0.01; (***, •••, ◊◊◊) for p < 0.001.

Figure 7

The viability assessment of human fibroblast MRC-5 and human melanoma MeWo cells after treatment with methanolic extracts (KE-Ex), 40% MeOH fraction (KE-Fr 40%), and acteoside (KE-Ref) from Kickxia elatine (L.) Dumort callus biomass for 24, 48, and 72 h presented as a line graph. The cell viability is expressed as a percentage of the nontreated control cells. The mean of three experiments ± SD is shown. Symbols of respective levels of significance (*,•, for p < 0.05; (**, ••, ◊◊) for p < 0.01; (***, •••, ◊◊◊) for p < 0.001.