Anti-cancer Properties of Myricetin Against HT-29 Colon Cancer Cells Through Regulation of RIPK1/RIPK3 Signaling Pathway

3.1. Cell Culture

HT-29 cell line was from the Iranian National and Genetic Center. The cells had grown in complete media and were maintained in a moist temperature incubator with 5% CO₂ and 37°C. After dissolving MCN in Dimethyl sulfoxide (DMSO), DMEM had used to dilute and obtain the desired concentration. A control group was exposed to only media, and the experimental groups were treated with three mM Necrostatine-1 (Nec-1, a necroptosis inhibitor), 50 μM MCN, and Nec-1 + MCN for 48 hours, respectively. Based on the MTT assay, the incubation time and concentration of MCN were selected (Figure 1). Nec-1 was also dissolved in 1% DMSO.

Figure 1.

The viability percentage of HT-29 cells in different concentrations and incubation times. Each assay has been done six times (4 replicates per assay), and the mean ± standard deviations have been indicated. * P < 0.05, ** P < 0.01, # P < 0.05; *, # and $ symbols indicate comparison to the control, and MCN50 groups.

3.2. Cell Viability Assay

MTT (dimethylthazolk 5-diphenyl tetrazolium bromide) test is applied to measure cellular metabolic activity for determining cell survival. It is a colorimetric method based on the cleavage of the tetrazolium ring of MTT by mitochondrial succinate dehydrogenase in living cells. Cells were exposed to MCN (0, 25, 50, 100, and 200 m) for 24, 48, and 72 hours. The cells were then incubated with MTT solution (0.5 mg/mL) for 3 hours, and the supernatants were replaced with DMSO (100 L). Finally, the absorbance was measured at 570 nm.

3.3. Annexin V / PI Apoptosis Assay

The cells were harvested in 6-well dishes (10⁶ cells/ well) and exposed to MCN or Nec-1 for 48 hours. Then, an Annexin V (FITC)/ PI (propidium iodide) kit (Invitrogen, USA) was used to determine the percentages of the dead or survival cells by flow cytometry. The FITC^-/PI^- cells were viable, while FITC⁺/PI^-, FITC⁺/PI⁺, and FITC^-/PI⁺ cells indicated early apoptosis, late apoptosis, and necrosis stages, respectively.

3.4. Real-Time PCR

An RNeasy kit was used to isolate the RNA of the cells (107 cells), and a cDNA kit was used to convert the RNA to cDNA. Two μL of the prepared cDNA amplified in a 25 μL PCR reaction contained SYBR Green and primers (Appendix 1 in Supplementary File). The following 45-cycle program was used to amplify the PCR: 95°C for 10 seconds, 95°C for 15 seconds, 60°C for 20 seconds, and 60°C for 20 seconds. The 2^-ΔΔCT method was applied to analyze the data.

3.5. Western Blotting Analysis

After treatment, HT-29 cells were washed and added to radioimmunoprecipitation (RIPA) lysis buffer. A BCA assay kit was used to determine the protein concentration within the cells. Lysate protein separated on 10% SDS-PAGE and transferred onto a PVDF membrane. The HT-29 cells were incubated with diluted primary antibodies (anti-RIPK1: ab178420; anti-RIPK3: ab222320; anti-MLKL: ab184718; anti-beta-Actin: ab115777) for 3 hours. The cells were then treated with horseradish peroxidase (HRP)-conjugated secondary antibody (ab205719) for 90 min. Proteins were detected using an ECL detection kit. Image J software was used to quantify the band density.

3.6. Statistical Analysis

3. Data were analyzed in SPSS (version 21.0) by one-way analysis of variance, followed by posthoc pairwise comparison with LSD or Kruskal-Wallis for non-parametric data. The P-values less than 0.05 were statistically significant.

4.1. Cell Viability

As illustrated in Figure 1, after 24 hours of incubation with various doses of MCN, no considerable decline in the viability of the HT-29 cells was observed. A significant decrease in the cell survival rate from 100% in control to 78.1%, 49.8%, and 42.8% in 25, 50, and 100 μM-treated cells were observed during 48 hours, respectively. No cell survival rate alterations occurred during 72 hours of incubation with 100 and 200 μM/mL MCN compared to the 48 hours. Cell viabilities at the concentration of 200 μM MCN during 48 and 72 hours were 23.7% and 25.8%, respectively, and hence were considered lethal concentrations (Figure 1). The half-maximal inhibitory concentration (IC50) results had used to determine the half-maximal concentration of MCN required to inhibit the growth of HT-29 cells within 48 hours. Treatment with MCN + Nec-1 significantly increased the survival of the HT-29 cells (Figure 2) compared to the only MCN-treated cells (P < 0.05).

Figure 2.

The viability percentage of HT-29 cells (mean ± SD). Each assay was done six times. * P < 0.05, ** P < 0.01, # P < 0.05, $ P < 0.05; *, # and $ indicate comparison to the control, Nec-1, and MCN groups, respectively.

4.2. Annexin V-FITC/ PI Assay

An annexin V/PI staining was performed to determine whether the MCN induced apoptosis or necrosis in HT-29 cells. In the untreated group, 98.9% of the cells were viable, 0.48% were in apoptosis, and 0.61% were in the necrosis stage. In comparison to the control, the proportion of viable cells decreased from 98.9 to 96.39%, 53.15%, and 81.1% in the Nec-1, MCN, and Nec-1 + MCN-treated cells, respectively. MCN slightly enhanced the percentage of apoptosis and the percentage of necrosis in HT-29 cells (75.7-fold, P < 0.001). Nec-1 + MCN caused a significant elevation in apoptosis (13.5-fold, P < 0.001), and a significant reduction in the necrosis (4.6-fold, P < 0.01) of the HT-29 cells compared to the MCN group (Figure 3).

Figure 3.

Flow cytometry results of Annexin V/PI staining (mean ± SD, n = 3). Lower left quadrant: Live cells, lower right quadrant: Early apoptosis, upper right quadrant: Late apoptosis, upper left quadrant: Necrotic cells. * P < 0.05, ** P < 0.01, *** P < 0.001, # P < 0.05, ## P < 0.01, ### P < 0.001, $ P < 0.01.

4.3. Real-Time PCR Assay

The expression of the RIPK1 gene was low in the Nec-1-exposed HT-29 cells. RIPK3 and MLKL expression in cells exposed to Nec-1 were unchanged compared to the control. MCN significantly enhanced the expression of MLKL, RIPK1, and RIPK3 compared to the control (P < 0.01). In the Nec-1 + MCN group, the mRNA expression of MLKL and RIPK3 genes was considerably lower than in the MCN-treated cells (Figure 4).

Figure 4.

mRNA expression of the different groups (mean ± SD, n = 5). *P < 0.05, ** P < 0.01, *** P < 0.001, # P < 0.05, ## P < 0.01, $ P < 0.05, $$ P < 0.001.

4.4. Western Blotting Analysis

In HT-29 cells treated with Nec-1, the RIPK1 protein was not expressed. The protein levels of MLKL and RIPK3 in the Nec-1-exposed HT-29 cells were not changed compared to the control.

MCN caused a significant elevation in the protein level of RIPK1, RIPK3, and MLKL compared to the control (P < 0.001). In the Nec-1 + MCN group, the protein expression of RIPK3 and MLKL was obviously lower than in the MCN-treated cells (P < 0.01) (Figure 5).

Figure 5.

Protein expression of HT-29 cells in different groups (mean ± SD, n = 3). Image J software was used to quantify the protein levels, and the cells normalized to β-actin band intensity. * P < 0.01, ** P < 0.001, # P < 0.01, ## P < 0.001, $ P < 0.01, $$ P < 0.001.

5.1. Conclusions

Overall, this study demonstrated that MCN inhibits colon cancer cell growth effectively. MCN causes high cytotoxicity in HT-29 cells due to the stimulation of necroptosis signaling.