The research evaluating the effect of 5-FU on the expression level of the SMAD4 gene and on apoptosis induction or DNA damage in colorectal cancer was conducted on a human colon cancer cell line (CACO-2) commercially purchased by Sigma Aldrich, Germany. The company provides a certificate of authenticity for the CACO-2 cell line, with the number of ECACC 86010202. The manufacturer’s data show, that the CACO-2 cells used for the research were originally derived from colon tissue obtained from a 72-year-old Caucasian male diagnosed with colorectal adenocarcinoma. The selected CACO-2 cell line was characterized for different molecular markers. Based on the data collected in the analysis, cells derived from the CACO-2 cell line showed no structural changes within the SMAD4 gene.
The CACO-2 cell line shows an adherent type of growth. The cells from the selected cell line were cultured in flasks with Eagle's Minimum Essential Medium supplemented with FBS and an antibiotic. The cells were grown in an incubator with a 5% CO2 atmosphere, at the temperature of 37°C and the appropriate air humidity. When confluence of the cells was 70–80%, the cell culture was passaged using trypsin.
Assessment of cell viability by MTT assay
The MTT test was performed to evaluate the 5-FU cytotoxicity effect on CACO-2 cells and to select an appropriate dose range of the studied drug. The assay was performed in a 96-well plate. Appropriately designed plates were seeded with a suspension of CACO-2 cells at a density of 1.5 x 104 cells / ml in a volume of 100 µl per well. Then, after 24hours the old medium was removed from the plate and the test compound dissolved in fresh culture medium was added to the wells in increasing concentrations. For this test, a 5-FU solution was used at the following concentrations: 0.1, 1, 5, 10, 100, 1000 µmol/l. Both plates were incubated for 24 hours and 48 hours of exposure to 5-FU. Following the end of incubation, the medium containing the test compound was removed and the cells were treated with a solution of MTT with a final concentration of 5 mg / ml. After a two-hour reaction at 37° C, the formed formazan crystals were dissolved with a 20% solution of sodium lauryl dodecyl sulphate (SDS) in 1M HCl. The plates were placed in the incubator overnight. At the last stage, the plates were read colorimetrically by measuring the absorbance at a wavelength of 570nm. Based on these data, the effect of 5-FU on cell viability was assessed and the concentrations of the test compound were selected, which was further used in the next stages of the experiment to assess the level of SMAD4 gene expression after exposure to the 5-FU.
Assessment of cell death by MUSE test kit
The effect of 5-FU on the initiation of apoptosis in CACO-2 cells was assessed with a Guava Muse Cell Analyzer flow cytometer using the Muse® Annexin V & Dead Cell Assay Kit. This method allows the detection of four cell populations, i.e. living cells, dead cells, and early and late apoptotic cells, based on binding of fluorescently labeled annexin V with phosphatidylserine (PS) located on the cell membrane. For the analysis, the cells of the CACO-2 line were cultured in 6-well plates. After a 24-hour incubation period, cells were treated with 5-FU solution in different concentrations, i.e. 0.1, 1, 5, 10, 100, and 1000 µmol/l and the exposure was carried out for 24hours. After this time, the cells were harvested from the surface of the wells in the plate using trypsin. The obtained cell suspensions in the culture medium were used for cytometer assay according to the manufacturer's instructions.
Assessment of DNA damage by MUSE test kit
An analysis of DNA damage through the ATM dependent pathway in the cell line CACO-2 under the influence of 5-FU was performed with a Guava Muse Cell Analyzer cytometer using a Flow Cellect Multi-Color DNA Damage Response reagent kit. Based on the test it is possible to differentiate the analyzed cells into four groups, i.e. cells without DNA damage, ATM activated cells, H2A.X activated cells and cells with double DNA strand breaks using fluorescent-based analysis. For the assay, CACO-2 cells were grown in 6-well plates and treated with 5-FU at concentrations of 0.1, 1, 5, 10, 100 and 1000 µmol/l. After a 24-hour exposure, cells were trypsinized from the plates into tubes and resuspended in culture medium, then centrifuged and washed with PBS (phosphate buffered saline) buffer solution. The obtained cell suspensions in PBS were used for the assay according to the manufacturer's instructions.
Assessment of SMAD4 gene expression in 5-FU treated CACO-2 cells:
Exposure of CACO-2 cells to 5-FU:
The 5-FU exposure procedure was performed in two 6-well plates. The first step was to apply a suspension of CACO-2 cells with a density of 1.5 x 104 cells/ml on a plates in a volume of 3 ml per well. The cell culture on the plates was carried out for 24hours. Subsequently, a series of dilutions of the test compound were prepared in antimicrobial-free culture medium. Having been seeded with cells, the prepared solutions of 5-FU at concentrations of 0.1, 1, 5, 10 and 100 µmol/l were applied to the plate. CACO-2 cells incubated with growth medium only and not treated with the test compound were used as a negative control. After the set exposure time (24 hours and 48 hours), the old medium containing the test substance was removed from the plates and the procedure of collecting cells from the plate using trypsin was performed. In the last stage, the test cell suspensions were taken from the wells on the plate into test tubes, centrifuged, washed twice with the buffer solution and finally suspended in PBS. The prepared cells treated with 5-FU were stored at -80°C until RNA isolation was performed.
RNA isolation from 5-FU-treated cells:
In order to evaluate the effect of 5-FU on the expression level of the SMAD4 gene in the CACO-2 cell line, isolation of RNA was performed using the column method with Genomic Midi Kit (A&A Biotechnology, Poland) in accordance with the manufacturer’s protocols. The concentration and purity of RNA obtained after isolation was assessed spectrophotometrically. RNA samples that had the ratio of the absorbance at 260/280 nm between of 1.8–2.0 were selected for further study.
Reverse transcription reaction
Total RNA obtained from the 5-FU treated cell line was subjected to a reverse transcription reaction to complementary DNA (cDNA) using High – Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, USA) according to the manufacturer’s protocol. The final RNA concentration of 0.005 µg/µl was determined for each sample. The presence of cDNA in the samples was checked by PCR for the GAPDH reference gene. Samples which confirmed the presence of 189 bp PCR product were stored at -20°C for further analysis.
Real – time PCR
Quantitative analysis of the expression level of SMAD4 mRNA as compared to the GAPDH reference gene was carried out using the Real-time PCR technique on a Bio-Rad apparatus (CFX Connect Real-Time PCR Detection System) in accordance with the manufacturer's protocol. The real-time reaction was performed with a non-specific fluorescent dye, using the iTaq Universal SYBR Green Supermix reagent kit (BioRad, USA). The composition of the reaction mixture for both genes included 5 µl of mix reagent, 0.25 µl of 10 µmol/l each primer solution, 1 µl of cDNA template and nuclease-free water to a final reaction volume of 10 µl. The sequences of the primers used for the reactions were GAPDH F 5’-TGGTATCGTGGAAGGACTCATGAC-3’; GAPDH R 5’-ATGCCAGTGAGCTTCCCGTTCAGC-3’; SMAD4 F 5’-GCCTGATCTTCACAAAAATG-3’; SMAD4 R 5’-GATCAATTCCAGGTGATACAAC-3’. Real-time PCR reactions for SMAD4 and GAPDH were run simultaneously, in separate tubes and in triplicate for each sample. A triplicate negative control amplification reaction was performed for each experiment. The thermal cycling conditions for the reaction were initial denaturation step at 95°C for three minutes, 39 cycles with two steps, i.e. denaturation at 95°C for 10 sec, annealing with elongation at 58°C for 30 sec. To confirm Real-time reaction specificity, the melting curve analysis for each amplification product was performed. The mean of the obtained Ct values for GAPDH and SMAD4 genes was calculated. To assess the relative level of SMAD4 expression the ΔΔCt method was used.