Milk thistle nano-micelle formulation promotes cell cycle arrest and apoptosis in hepatocellular carcinoma cells through modulating miR-155-3p /SOCS2 /PHLDA1 signaling axis

Abstract

Background: Hepatocellular Carcinoma (HCC) is a prevalent form of liver cancer that causes significant mortality in numerous individuals worldwide. This study compared the effects of milk thistle (MT) and nano-milk thistle (N-MT) on the expression of the genes that participate in apoptosis and cell cycle pathways in Huh-7 and HepG2 cells.

Methods: IC50 values of MT and N-MT were determined using the MTT assay. Huh-7 and HepG2 cell lines (containing mutant and wild-type TP53 gene, respectively) were incubated with MT and N-MT for 24h and 48h and the impact of MT and N-MT on the proliferation of these cell lines was evaluated through a comparative analysis. Cell cycle and apoptosis were assessed by flow cytometry after 24h and 48h treatment in the cell lines mentioned. Real-time PCR was used to analyze miR-155-3p, PHLDA1, SOCS2, TP53, P21, BAX, and BCL-2 expression in the cell lines that were being treated.

Results: N-MT reduces cancer cell growth in a time and concentration-dependent manner, which is more toxic compared to MT. Huh-7 was observed to have IC50 values of 2.35 and 1.7 μg/ml at 24h and 48h, and HepG2 was observed to have IC50 values of 3.4 and 2.6 μg/ml at 24 and 48h, respectively. N-MT arrested Huh-7 and HepG2 cells in the Sub-G1 phase and induced apoptosis. N-MT led to a marked reduction in the expression of miR-155-3p and BCL-2 after 24h and 48h treatments. Conversely, PHLDA1, SOCS2, BAX, and P21 were upregulated in the treated cells compared to untreated cells, which suggests that milk thistle has the potential to regulate these genes. N-MT reduced the expression of TP53 in Huh-7 cells after mentioned time points, while there was a significant increase in the expression of the TP53 gene in HepG2 cells. No gene expression changes were observed in MT-treated cells after 24h and 48h.

Conclusion: N-MT can regulate cancer cell death by arresting cell cycle and inducing apoptosis. This occurs through the alteration of apoptotic genes expression. A reduction in the expression of miR-155-3p and increase in the expression of SOCS2 and PHLDA1 after N-MT treatment showed the correlation between miR-155-3p and PHLDA1/SOCS2 found in bioinformatics analysis. While N-MT increased TP53 expression in HepG2, reduced it in Huh-7. The findings indicate that N-MT can function intelligently in cancer cells and can be a helpful complement to cancer treatment.

Keywords: Apoptosis; Cell cycle arrest; Nano-milk thistle; Noncoding RNA.

Fig. 1

Effect of MT and N-MT on cell viability. A, B Huh-7 cells were treated with MT and N-MT and their viability was examined by MTT assay after 24h and 48h. C, D HepG2 cells were treated with MT and N-MT and their viability was examined by MTT assay after 24h and 48h. E HDF cells were treated with N-MT and their viability was examined by MTT assay after 24h and 48h. N-MT induces apoptosis in Huh-7 and HepG2 cells in a time- and dose-dependent manner, while the effect of MT extract on the induction of apoptosis in these cells is much lower than that of N-MT. N-MT has no impact on normal cells (fibroblasts) as well. ∗ P < 0.05, ∗  ∗ P < 0.01, ∗  ∗  ∗ P < 0.001, ∗  ∗  ∗  ∗ P < 0.0001

Fig. 2

Predicted LINC01093 and 3’ UTR of SOCS2 and PHLDA1 matched with the seed sequence of miR-155-3p

Fig. 3

Real-time PCR analysis of three cancer-associated genes after treating the cells with N-MT and MT in Huh-7 and HepG2 cells. Huh-7 cells were treated with 2 and 1.5 µg/mL N-MT and MT for 24h and 48h, respectively. HepG2 cells were treated with 2.3 and 3.1 µg/mL N-MT and MT for 24h and 48h, respectively. A, B miR-155-3p, C, D PHLDA1, E, F SOCS2. The mRNA expression of the cancer-related genes was evaluated by the optimized real-time PCR method at 24h and 48h after treatment. The results are presented as the mean mRNA expression fold changes compared to the untreated. The analyses indicated that N-MT caused PHLDA1 and SOCS2 mRNA level upregulation, while miR-155-3p gene expression decreased in both cell lines compared to the untreated cells. ∗ P < 0.05, ∗  ∗ P < 0.01, ∗  ∗  ∗ P < 0.001, ∗  ∗  ∗  ∗ P < 0.0001, ns: non-significant

Fig. 4

Real-time PCR analysis of the TP53 gene after treating the cells with N-MT and MT in Huh-7 and HepG2 cells. Huh-7 cells were treated with 2 and 1.5 µg/mL N-MT and MT for 24h and 48h, respectively. HepG2 cells were treated with 2.3 and 3.1 µg/mL N-MT and MT for24h and 48h, respectively. A Huh-7, B HepG2. The mRNA expression of the cancer-related genes was evaluated by the optimized real-time PCR method at 24h and 48h after treatment. The results are presented as the mean mRNA expression fold changes compared to the untreated cells. ∗ P < 0.05, ∗  ∗ P < 0.01, ∗  ∗  ∗ P < 0.001, ns: non-significant

Fig. 5

Real-time PCR analysis of three apoptosis-related genes after treating the cells with N-MT and MT in Huh-7 and HepG2 cells. Huh-7 cells were treated with 2 and 1.5 µg/mL N-MT and MT for 24h and 48h, respectively. HepG2 cells were treated with 2.3 and 3.1 µg/mL N-MT and MT for24h and 48h, respectively. A, B P21, (C, D) BAX, (E, F) BCL-2. The mRNA expression of the apoptosis-related genes was evaluated by the optimized real-time PCR method at 24h and 48h after treatment. The results are presented as the mean mRNA expression fold changes compared to the untreated. The analyses indicated that N-MT caused P21 and BAX mRNA level upregulation, while BCL-2 gene expression significantly decreased in both cell lines compared to the untreated cells. ∗ P < 0.05, ∗  ∗ P < 0.01, ∗  ∗  ∗ P < 0.001, ns: non-significant

Fig. 6

Effect of N-MT on cell cycle in Huh-7 cells, using flow cytometry. Huh-7 cells were treated with 2 and 1.5 µg/mL nano missile encapsulated milk thistle for 24h and 48h, respectively. Panel (A) Shows the population of untreated cells in Sub-G1/G1, S and G2/M stages. Panel (B, C) Show the population of N-MT-treated Huh-7 cells in Sub-G1/G1, S and G2/M stages at two different time points (24h and 48h) after treatment. Panel (D) Represents the percentage of the Huh-7 cells in sub-G1/G1 stage after 24h and 48h treatment compared to the control group Sub-G1/G1 cell proportion has increased due to the effect of N-MT in a time-dependent manner. ∗ P < 0.05, ∗  ∗ P < 0.01

Fig. 7

Effect of N-MT on cell cycle in HepG2 cells, using flow cytometry. HepG2 cells were treated with 2.3 and 3.1 µg/mL nano missile encapsulated milk thistle at 24h and 48h, respectively. Panel (A) Shows the population of untreated cells in Sub-G1/G1, S and G2/M stages. Panel (B, C) Show the population of N-MT-treated HepG2 cells in Sub-G1/G1, S and G2/M stages at two different time points (24h and 48h) after treatment. Panel (D) Represents the percentage of the HepG2 cells in sub-G1/G1 stage after 24h and 48h treatment compared to the control group. Sub-G1/G1 cell proportion has increased due to the effect of N-MT in a time-dependent manner. ∗ P < 0.05, ∗  ∗ P < 0.01

Fig. 8

Cellular apoptosis analysis N-MT-treated Huh-7 cells. Huh-7 cells were treated by 2 and 1.5 µg/mL nano missile encapsulated milk thistle, and then, the apoptotic population was measured by the Annexin V-PI assay, using flow cytometry at 24h and 48h. A Flow cytometry of the apoptosis for untreated cells,(B, C) Flow cytometry of the apoptosis for N-MT-treated Huh-7 cells at two different time points (24h and 48h) after treatment, and (D) Apoptotic cell percentage of total cells was increased at 24h, and 48h in Huh-7 cell line. ∗  ∗ P < 0.01, ∗  ∗  ∗ P < 0.001

Fig. 9

Cellular apoptosis analysis in N-MT-treated HepG2 cells. HepG2 cells were treated by 2.3 and 3.1 µg/mL nano missile encapsulated milk thistle, and then, the apoptotic population was measured by the Annexin V-PI assay, using flow cytometry at 24h and 48h. A Flow cytometry of the apoptosis for untreated cells, B, C Flow cytometry of the apoptosis for N-MT-treated HepG2 cells at two different time points (24h and 48h) after treatment, and D Apoptotic cell percentage of total cells was increased at 24h, and 48h in HepG2 cell line. ∗  ∗ P < 0.01, ∗  ∗  ∗ P < 0.001

Fig. 10

A schematic representation of the predicted ceRNA pathway in a cancer cell treated with N-MT. As can be seen, N-MT-treated cancer cells witnessed an alteration in various genes expression that eventually leads to apoptosis of cancer cells. LINC01093 has been predicted to have an association with miR-155-3p which is decreased and result in the SOCS2 and PHLDA1 increment. (This figure was drawn by the BioRender site (https://app.biorender.com)