In vitro Cytotoxicity Evaluation of Dandelion Root Ethanol Extract on PANC-1 Cell Line

: A challenging diagnosis is the outcome of pancreatic cancer's aggressivity and malignant character. This type of cancer is mostly detected in the metastatic stage. Current treatment options have various side effects, result in the survival rates and quality of life of patients are extremely decreased. Dandelion (Taraxacum officinale (L.) Weber ex F.H. Wigg.) is one of the natural products that has the potential to be therapeutic against a wide range of diseases. This study aimed to evaluate the potential of ethanol extract of dandelion root (DRE) as an alternative anticancer agent. Based on this purpose, milk-rich dandelion roots were collected, cut into small pieces, and then extracted using Soxhlet in the presence of 70% ethanol. The cytotoxic effect of the DRE at certain doses (10, 5, and 2.5 mg/mL) was determined by the Microtetrazolium (MTT) assay for 24, 48, and 72 hours, and half-maximal inhibitory concentration (IC)50 values for these incubation periods were found to be >10 mg/mL, 6.80 mg/mL and 6 mg/mL (p<0.001), respectively. As a result, DRE may reduce cell viability when applied to aggressive pancreatic cancer cells and may have the potential to be an alternative anticancer agent. This potential can be clarified with further studies.


INTRODUCTION
Pancreatic cancer is one of the most aggressive forms of cancer.It is a fatal disease that develops when normal pancreatic cells proliferate and grow out of control.It occurs due to mutations in various cancer-related genes, including tumor suppressor genes, cell cycle genes, apoptosis, and genome repair genes in germ cells and somatic cells. 1,2This deadly form of cancer does not show early symptoms and has the potential to invade surrounding tissues and organs rapidly. 3When current treatment strategies are evaluated, surgical intervention is the primary option for treating pancreatic cancer.However, for patients who are unable to undergo a surgical intervention due to the common late diagnosis of pancreatic cancer and their metastatic ability, chemotherapy, radiotherapy, and immunotherapy constitute other treatment options.Therefore, for patients with pancreatic cancer who have few treatment options and an aggressive progression rate, alternative treatment approaches are crucial. 4,5ue to their chemical diversity and reservoirs of bioactive compounds with therapeutic potential, natural products are part of research on alternative treatments.][14][15] It has been reported in various studies that ethanol extract has a higher content of bioactive molecules than water extract. 16Although the cytotoxicity of the water extract from the dried dandelion root on the pancreatic cancer cell line has already been established, 11 no research has been encountered in the literature about the cytotoxic impact of the ethanol extract made from the fresh dandelion root, which contains more bioactive molecules, on the PANC-1 cancer cell line.Therefore, in this study, the cytotoxicity of a 70% ethanol | 67 extract obtained from dandelion root against the pancreatic cancer cell line PANC-1 was determined by the MTT method.

MATERIAL AND METHOD Ethanol Extraction of Dandelion Roots
The dandelion plant was collected from the parks and gardens in Konya's Meram district in May without damaging the roots.The roots were cleaned of soil with dH2O, washed, had water removed after washing, and cut into small pieces of approximately 0.5 mm in size.25 g of sliced roots were placed in the Soxhlet column, and extraction was performed in the presence of 110 mL of 70% ethanol.Furthermore, the solvent was evaporated in a rotary evaporator at 60 RPM and 86 °C.

Cell Culture
In this study, the PANC-1 (human epithelioid carcinoma) cell line was used.To cultivate the cells, a completed DMEM medium with 10% Fetal Bovine Serum and 1% penicillin/streptomycin (100 U/ml penicillin and 100 μg/ml streptomycin) was used for culturing of the cells.Sterile laminar airflow cabinets were utilized to conduct culture processes.The cells were incubated at 37°C with 5% CO2 in an incubator.The old medium was replaced, and a new medium was added every 2-3 days until the cells covered the surface.When the cells covered the flask surface by 80-90%, the passage was performed with trypsin-EDTA.An inverted microscope was used to perform growth control and contamination checks on the cells.

Cytotoxicity Analysis
Approximately 10 4 cells in 200 µl of growth media were put into each well of microplate for the MTT assay.The well plate was kept in an incubator at 37°C and 5% CO2 for 24h.At the end of this period, the old medium was removed, and the cells in the wells were treated with DRE (10-2.5 mg/mL) at concentrations determined by taking into account previously performed studies. 13,17Each dose was administered in triplicate.During dosing, care was taken to keep the solvent DMSO ratio constant at 0.1% for each dose application. 18egative control cells were treated with a medium containing DMSO at the same concentration.After application, cells were incubated in a 37°C, 5% CO2 incubator for 24, 48 and 72 hours.At the end of the periods, the media was removed, and 20 µl of Thiazolyl Blue Tetrazolium Bromide (MTT) solution was added to the wells and incubated for 3 hours at 37°C, 5% CO2.At the end of the 3 hours, MTT dye was removed from the wells, and 200 µl DMSO was added and shaken in an orbital mixer for 1 hour.After incubation, the absorbance of the wells was measured at a wavelength of 570 nm on a microplate reader.Cell viabilities were calculated according to the formula stated below. 19

Statistical Analysis
Results were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison Test.Statistical analyses obtained in this study were performed using GraphPad Prism version 8.0.2 software (GraphPad Software, Inc., San Diego, California, United States).

RESULT
In this study, the MTT method was used to evaluate the anticancer activity of DRE against the pancreatic cancer cell line PANC-1 at various doses and exposure times.When DRE was used at a concentration of 10 mg/mL was applied to PANC-1 cells, it inhibited cell growth by 26%, 72%, and 84% for 24, 48, and 72 hours (Figure 1 and Figure 2A).The evaluation was carried out for three separate incubation periods, 24, 48, and 72 hours, and IC50 values for these incubation periods were found to be >10 mg/mL, 6.80 mg/mL and 6 mg/mL (p<0.001),respectively (Figure 2B).

DISCUSSION
This study investigated the cytotoxic effects of ethanol extract obtained from Dandelion root on PANC-1, human pancreatic cancer cells.The treatment of PANC-1 cells with 10 mg/mL of DRE produced the maximum inhibition rate, and the lowest IC50 value was reached during the 72-hour treatment period.As a result, cytotoxic activity increased with longer exposure periods and higher DRE concentrations.It shows that the DRE has a cytotoxic effect against pancreatic cancer and exhibits anticancer activity.When these findings are contrasted with prior research by Ovadje et al. (2012), they agree with the IC50 values derived from the dosages of water extract used. 11When different literature data are evaluated together with our results, the anticancer effects of extracts made with different solvents acquired from dandelion roots confirm each other.When different literature data are evaluated collectively with our results, the anticancer effects of extracts made with different solvents were obtained from dandelion roots that confirmed each other.When different literature data are evaluated collectively with our results, the anticancer effects of extracts made with different solvents were obtained from dandelion roots that confirmed each other.In one of these investigations, Rehman et al. (2017) documented the efficaciousness of dandelion root methanol extract on HepG2, HCT116, and MCF7 cancer cell lines. 20In a different study, the anti-cancer properties of dandelion root extract were assessed in gastric cancer cell lines (SGC7901 and BGC823), and the results showed that the extract inhibited the cells' ability to proliferate and migrate. 21Further studies are needed to analyze the compounds found in dandelion roots and evaluate their selective anticancer potential.However, current results are established from the literature, and the data reported in this study revealed the potential of dandelion root extracts as cytotoxic anticancer agents.

CONCLUSION
The findings on the cytotoxic concentration range of ethanol extract obtained from fresh latex-filled dandelion roots presented here demonstrated that even on highly aggressive pancreatic cancer cell lines, it had cytotoxic effects.These results might support the potential of dandelion root extract as an anticancer agent.
Therefore, we suppose that with more research and the ability to do both in vitro and in vivo testing, the bioactive compounds in the content can be successfully isolated for therapeutic use.Furthermore, it is important to remember that every cell type has a unique reaction to various extracts made by using various solvents and that distinct cancer cells and healthy cells may react differently to each extract.Based on this reason, analyzing data from various cancer cell lines and extracts made using various solvents is crucial to the development of anticancer drugs.

Figure 1 .Figure 2 .
Figure 1.Cell Morphology after treatment with the DRE for 24 h, 48 h, and 72 h