Apoptosis induction by siRNA targeting integrin-β1 and regorafenib/DDAB-mPEG-PCL hybrid nanoparticles in regorafenib-resistant colon cancer cells

Abstract

Colorectal cancer (CRC) is regarded as the third most common cancer worldwide. Although Regorafenib as a receptor tyrosine kinase inhibitor (RTKI) disrupts tumor growth and angiogenesis in metastatic CRC (mCRC) patients, drug resistance leads to poor prognosis and survival. Integrin-β1 overexpression has been proposed to be the major player in this regard. Herein, the Regorafenib-resistant human colon cancer cell line (SW-48) was induced, and the Integrin-β1 gene expression, as well as apoptosis, was assessed through the combination of small interfering RNA (siRNA) targeting Integrin-β1 and Regorafenib/Dimethyldioctadecylammonium bromide (DDAB)-methoxy poly (ethylene glycol) (mPEG)-poly-ε-caprolactone (PCL) hybrid nanoparticles (HNPs). In the current study, Regorafenib-resistant SW-48 cell line was generated in which the Regorafenib half-maximal inhibitory concentration (IC₅₀) for non-resistant and resistant cells was 13.5±1.5 µM and 55.1±0.8 µM, respectively. The results of DLS also demonstrated that the size and the charge of the HNPs were equal to 66.56±0.5 nm and +29.5±1.2 mv, respectively. In addition, the Integrin-β1 gene expression was significantly higher in resistant cells than in non-resistant ones (P<0.05). The siRNA/HNP complexes in combination with Regorafenib/HNPs were accordingly identified as the most effective treatment to decrease the Integrin-β1 gene expression and to enhance the apoptosis rate in resistant cells (P<0.001). Overall, the study indicated that combination therapy using siRNA/HNP and Regorafenib/HNPs complex could down-regulate the Integrin-β1 gene expression and consequently trigger apoptosis, and this may potentially induce drug sensitivity.

Keywords: Colorectal cancer; SiRNA; apoptosis; integrin-β1; lipid-polymer hybrid nanoparticle; regorafenib.

Figure 1

Cell survival percentage in the SW-48 cell line at the end of each cycle using the MTT method. The cells were treated for four cycles by Regorafenib, and then the MTT assay was conducted. They experienced a high rate of mortality in the early cycles, but adapted to the environment and exhibited more viability at the end of cycles 3 and 4. According to the Tukey’s post-hoc test of multiple comparisons, significant differences were observed between each cycle, cycle 1 and the non-resistant group. **P=0.0017, ***P<0.0001.

Figure 2

Inhibitory effect of Regorafenib on the cell viability of non-resistant SW-48 cells in a dose-dependent manner. Cell viability was measured using the MTT method by incubating the cells to the increasing doses of Regorafenib (0-20 μM) for 24 hours. The IC₅₀ was 13.5±1.5 μM in non-resistant cells.

Figure 3

Inhibitory effect of Regorafenib on the cell viability of resistant SW-48 cells in a dose-dependent manner. Cell viability was measured using the MTT method by incubating the cells to the increasing doses of Regorafenib (0-60 μM) for 24 hours. The IC₅₀ was 55.1±0.84 μM in resistant cells.

Figure 4

Size distribution (A) and zeta potential (B) of DDAB-mPEG-PCL HNPs.

Figure 5

Size distribution (A) and zeta potential (B) of Regorafenib loaded HNPs.

Figure 6

N/P ratios of 0-60 on the agarose gel to determine the optimum N/P ratio for maximum binding of siRNA on HNPs’ surface. The N/P ratio of 20 was selected as the best N/P ratio.

Figure 7

Integrin-β1 gene expression levels in resistant and non-resistant SW-48 cell lines. Integrin-β1 mRNA fold change showed approximately 1.8 times higher value than that of the non-resistant one, which was statistically significant (P<0.05).

Figure 8

Comparison of the Integrin-β1 gene expression level in different treatment groups of resistant SW-48 cells. The use of free siRNA did not significantly affect Integrin-β1 gene expression (A). Significant differences were observed in the subgroups of (B-D) compared to the untreated control group. (E) Comparison of the effect of Regorafenib-loaded HNPs and free Regorafenib on the Integrin-β1 gene expression. HNPs and siRNA negative control/HNPs were used as controls. **P<0.01, ***P<0.001.

Figure 9

Intragroup comparison of the Integrin-β1 gene expression in resistant SW-48 cells. Comparison of the effect of 100 μΜ (A), 50 μΜ (B) and 25 μΜ (C) doses of siRNA when using alone, siRNA/HNP complexes, combination of D40 and siRNA/HNPs, as well as NPD40 and siRNA/HNPs. *P<0.05, **P<0.01, ***P<0.001.

Figure 10

Effect of Integrin-β1 siRNA 100 (A), siRNA-HNP 100 (B), D40+siRNA-HNP 100 (C), and NPD40+siRNA-HNP 100 (D) on the apoptosis rate of resistant SW-48 cells detected by FC. Q1, Q2, Q3, and Q4 quadrants represent necrotic, early apoptotic, late apoptotic, and normal cells, respectively. (E) show the control group (i.e., untreated group).

Figure 11

Comparison of the apoptosis rate in different treatment groups. The apoptosis assay was conducted using FC after being treated with different formulations of Integrin-β1 siRNA. Both early apoptotic (Annexin V⁺/PI^-) and late apoptotic (Annexin V⁺/PI⁺) cells were considered the determinants of cell death.