INTRODUCTION:

Artemisia vulgaris L is a member of the Asteraceae family's largest genus. Because of its adaptability, this herb is native to Europe and is rapidly expanding throughout the world^1-3. This herb was widely used in traditional medicine to boost the immune system, inhibit the invasion of germs, viruses, and fungi, and prevent tumour growth^4-6. More than 600 secondary metabolites have been found in the genus Artemisia L to far, with a variety of important bio-effects^7-11. Preliminary scientific research established that A. vulgaris has antioxidant, antibacterial, anti-inflammatory, and anti-cancer properties^12-19. At low concentrations, methanol extract of this herbal has been shown to trigger cancer cell death in HepG2, MCF-7, Hela, HL-60, and other cell lines^17,20-24. The potent active components identified included yomogin; 1,2,3,4-diepoxy-11(13)-eudesmen-12,8-olide and artemisinin, the latter of which is well-known as a life-saving chemical that has saved millions of people from malaria all over the world^25-27.

Tu Youyou received the Nobel Prize in Physiology or Medicine in 2015 for her work on artemisinin, which led scientists to pay attention to the Artemisia annua²⁸.

According to statistics on 36 different varieties of cancer from 185 nations and territories in 2020, cancer is one of the most important health crises confronting the entire world, with 19.3 million new cancer cases and more than 10 million fatalities²⁹. Leukaemia is the 15th most prevalent disease in terms of new occurrences, with 474,519 cases identified in 2020 (about 2.50 percent of total new cases) and accounting for 311,594 deaths globally (a rate of 3.10 percent)³⁰. Differences in medical levels and facilities between developed and developing countries resulted in differences in leukaemia patient survival rates. For example, in developed countries, more than 90% of patients with acute lymphoblastic leukemia (ALL) were healed, whereas it was 40% to 70% in low and middle-income countries^31,32. The emergence of SARS-CoV-2 triggered a worldwide COVID-19 epidemic, resulting in significant loss of life and economic upheaval^33,34. Cancer research has been significantly hampered as a result³⁵. The purpose of this study was to investigate the cytotoxic effect of A. vulgaris methanol extract (MetAV) on leukaemia cell lines (acute myeloid leukaemia - AML) containing the ETV6–NTRK3 fusion gene, which pointed the way for future in-depth research and AML curative therapy applications.

MATERIALS AND METHODS:

Plant materials and sample extraction preparation:

Artemisia vulgaris L. was collected and identified as previously²⁴. Cell lines from AML patients were verified as ETV6–NTRK3 positive, including IMS-M2 and MO-91^36-38. Stable expressing ETV6–NTRK3 BaF3 cell: BaF3-CFS was transfected with the ETV6–NTRK3 form congenital fibrosarcoma^39,40. The normal kidney tissue is derived from Vero The plant's aboveground herbaceous section was harvested, cleaned with distilled water, and drained before being properly dried in an oven at 40°C. The herbal powder was created by grinding the herbs in a blender. 30 grammes of herbal powder were macerated in an Erlenmeyer flask with a pure methanol solvent that submerged the powder by 1-2 cm. The constantly shaken mixture was filtered using Whatman paper every 24 hours for 5 times to obtain the filtrate. Rotovating the recovered filtrate yielded a crude methanol extract⁴¹. By soaking the crude extract in DMSO and passing the solution through 0.45- and 0.22 µm filters, a 200 mg/ml stock solution of the extract was produced. All of the procedures were carried out in the Biological Safety Cabinet. The extract was kept at -20°C until it was used.

Cell culture preparation:

The cells were cultured in Roswell Park Memorial Institute 1640 medium (Sigma-Aldrich) supplemented with 10% foetal bovine serum (Thermofisher Scientific), 100 IU/mL penicillin, and 0.1 mg/mL streptomycin (Sigma-Aldrich) in humidified incubator of 5% continuously loading CO₂ at 37^oC.

Cytotoxic assay:

The cell lines were seeded in 6-well plates with or without treatment at various concentrations of MetAV and incubated for 48 hours at 37°C with 5% CO2 for 48 hours. The well that did not receive the treatment additive served as the control. The extract's lethality was determined using the Trypan blue staining technique⁴². A volume of 20µL of the cell suspension was mixed with 20µL of 0.4% Trypan Blue (Merck). Cell membrane breakdown in dead cells allows dye to enter the cell and render it blue, but survival cells with intact cell membranes are not colored, resulting in clear cells⁴³. The number of living cells under treatment was determined by manually counting cells with a hemocytometer. The difference in the percentage of living cells between the treated and control groups reflected cell viability during treatment.

The effects of MetAV were evaluated in a dosage-dependent manner by assessing the extract at different concentrations (6.25; 12.5; 25; 50; and 100 µg/ml) at a cell starting density of 10⁵ cells/ml. The inhibitory concentration at half-maximal (IC50) was determined. The extract's effect at IC50 was further studied at varied starting cell densities (from 10⁴ to 10⁶cells/ml).

Data analysis:

The experiments were performed in independent triplicates. The data was statistically analysed using GraphPad Prism version 9.0.0. The results were shown as Means ± Standard deviation (SD). Nonlinear regression analysis was carried out to calculate the IC50. The One-way ANOVA and the post-hoc Tukey comparison test was performed for determining the statistically significant difference between the experimental groups. Significant differences were accepted at a p-value < 0.05 and noted as (*); (**); (***); and (****) for p-values that were less than 0.0332; 0.021; 0.0001; and 0.0002, respectively.

RESULTS:

Figure 1A represents the effect of MetAV on leukaemia cell lines. The results demonstrated that the extract had a devastating effect on cells expressing the ETV6–NTRK3 fusion gene at concentrations less than 100 µg/ml. In comparison to the leukaemia group, the effect of MetAV on the epithelial kidney cell line Vero was less effective (Figure B).

Figure 1: Cytotoxicity of MetAV on leukemia cell (A) and Vero cell (B)

The nonlinear regression analysis revealed that MetAV's IC50 values on the cell lines were 26.98 ± 2.25 , 21.85 ± 0.92, 18.96 ± 1.70, and 153.15 ± 3.63 (µg/ml) for IMS-M2; MO-91; BaF3-CFS and Vero, respectively. To compare the effects of MetAV on the proliferation of four cell lines, a one-way ANOVA was used. There was a significant effect of the extract on cell viability among the four cell lines, F (3) = 2331, p-value <0.0001. Post hoc test comparison, Tukey test, indicated that Vero cell was the least affected and BaF3-CFS was the most affected based on the mean score; however, the effect on MO-91 did not differ significantly from that on IMS-M2 and BaF3-CFS.

Figure 2: Cell morphology monitoring with and without MetAV

The selective index (SI) is defined as the ratio of the concentration making toxicity against its biological function of the valuated extract⁴⁴. The IC50 values of the extract from the Vero cell line were divided by the leukaemia cells to arrive at the SI values. IMS-M2 had a SI of 5.67; MO-91 had a SI of 7.01; and BaF3-CFS had a SI of 8.08. All of the SI values recorded were larger than 3.

An inverted light microscope with a magnification of 10X was used to observe the morphological changes in the cells in addition to the cytotoxicity test. The morphology of the cells in the treatment and control groups was examined, and comparisons were made between the two groups (Figure 2). The finding revealed some differences in cell size, appearance, and volume between two groups. In the treated wells, there were dead cells, cell shrinkage, blebbing membranes, and cell debris. The extract was found to cause greater cell death at increasing concentrations.

Various initial cell seeding densities were tested in order to see if cell density had any effect on the extract's efficacy. It was suggested that shielding and dilution at high cell densities might affect the effectiveness of the extract. The results of the extract impact at the IC50 were shown in detail in figure 3. The more concentrated of cell denseness, the cell viability increased for both of MO-91 and IMS-M2. A two-way ANOVA was performed to analyse the effects of the extract on the cell at different initial cell seeding densities. The results revealed that there was a statistically significant interaction among the effects of the extract on cells at different densities (F (4,8) = 270.9, p-value <0.0001). Thus, the density of the initially seeded cells had an impact on the extract's ability to influence cell growth.

Figure 3: The impact of initial cell density on the effects of MetAV

Moreover, a two-way ANOVA was performed to analyse the cell viability under the impact of the extract on MO-91 and IMS-M2 on several initial cell seeding densities and revealed that there was a statistically significant interaction between the effects of the extract on MO-91 and IMS-M2 (F (1,2) = 178.5, p-value = 0.0053). Šídák's multiple comparisons post-hoc test indicated that there was a significant difference in cell viability of MO-91 and IMS-M2 in each initial seeding density (P-values were 0.0013, 0.0002, < 0.0001, and < 0.0001 for the densities of 5x10⁴, 10⁵, 5x10⁵, and 10⁶, respectively). The results showed that the A. vulgaris methanol extract had a more aggravating effect on IMS-M2 than on MO-91, which was in slight conflict with the previous IC50 result.

DISCUSSION:

Recently, in a screening study, the methanol extract of A. vulgaris exhibited strong effects on leukaemia cell lines consisting of TCC-Y (IC50 = 45.87 ± 3.49 µg/ml), TCC-S (IC50 = 22.89 ± 0.68 µg/ml), KOPB-26 (IC50 = 22.00 ± 1.33 µg/ml), HL-60 (IC50 = 29.36 ± 2.26 µg/ml) and Ku-812 (IC50 = 18.07 ± 1.64 µg/ml)²⁴. IC50 values less than 30 µg/ml were judged highly cytotoxic, whereas IC50 values greater than 100 µg/ml were classified as non-active by the American National Cancer Institute (NCI)^45,46. In addition, the SI value shows the possible application of medicinal herbs by reflecting the balance between toxicity and bioactivity⁴⁷. Cell lines that were evaluated had SI values greater than three, which is a good sign for further research⁴⁸. The effect of the extract was stronger on the BaF3 cell line than on the clinical cell lines IMS-M2 and MO-91. Compared to the clinical cell lines IMS-M2 and MO-91, the BaF3 cell line was more responsive to the extract's effects. For the original BaF3 cell line, IL3 signaling was necessary for proliferation and development in mice B-cells⁴⁹. The survival signalling mechanism for BaF3-CFS was the transgenic production of the fusion protein Tel/TrkC, which assisted the cell in self-activating the proliferation pathway^39,40. In other words, MetAV showed the potential for comprehensive study and wide use in leukemia-related ETV6–NTRK3 fusion gene research.

Cell shrinkage and volume depletion were early indicators of the apoptosis pathway, which was observed during cell observation⁵⁰. Evidence suggests that A.vulgaris, by raising Apaf-1 expression preserving Bak, and Bcl-XL expression while decreasing Bcl-2 translation, induces cell death in HL-60 cells via the intracellular apoptotic pathway⁵¹. A variety of molecular targets, including pro-caspase, Cox-2, PARP, the Bcl family, and CD95L, were found to be sensitive to the Artemisia genus' ability to decrease cell proliferation⁵².

In vitro experiments rely on the initial cell seeding density^53,54. The results revealed that the higher the cell density, the lesser the extract's potency. Because of variances in chromosomal aberrations and signaling pathways, the extract's effect on the two clinical cell lines was unequal^36-38.

CONCLUSION:

In conclusion, MetAV demonstrated strong cytotoxicity against ETV6–NTRK3 fusion gene-related leukaemia cell lines while being non-toxic to normal cell lines. The extract's effect on leukaemia cells was discovered to be dosage and cell density dependent. The morphological changes in cells reflected the apoptosis-activating mechanisms that must be addressed in this herbal study.

COMPETING INTEREST:

The authors declare that they have no competing interests.

ACKNOWLEDGMENTS:

This research is funded by Thu Dau Mot University under grant number DT.21.1-059.

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Received on 24.10.2022 Modified on 12.12.2022

Research J. Pharm. and Tech 2023; 16(8):3825-3829.

DOI: 10.52711/0974-360X.2023.00631