131I-C19 Iodide Radioisotope and Synthetic I-C19 Compounds as K-Ras4B-PDE6δ Inhibitors: A Novel Approach against Colorectal Cancer-Biological Characterization, Biokinetics and Dosimetry

Abstract

In 40-50% of colorectal cancer (CRC) cases, K-Ras gene mutations occur, which induce the expression of the K-Ras4B oncogenic isoform. K-Ras4B is transported by phosphodiesterase-6δ (PDE6δ) to the plasma membrane, where the K-Ras4B-PDE6δ complex dissociates and K-Ras4B, coupled to the plasma membrane, activates signaling pathways that favor cancer aggressiveness. Thus, the inhibition of the K-Ras4B-PDE6δ dissociation using specific small molecules could be a new strategy for the treatment of patients with CRC. This research aimed to perform a preclinical proof-of-concept and a therapeutic potential evaluation of the synthetic I-C19 and ¹³¹I-C19 compounds as inhibitors of the K-Ras4B-PDE6δ dissociation. Molecular docking and molecular dynamics simulations were performed to estimate the binding affinity and the anchorage sites of I-C19 in K-Ras4B-PDE6δ. K-Ras4B signaling pathways were assessed in HCT116, LoVo and SW620 colorectal cancer cells after I-C19 treatment. Two murine colorectal cancer models were used to evaluate the I-C19 therapeutic effect. The in vivo biokinetic profiles of I-C19 and ¹³¹I-C19 and the tumor radiation dose were also estimated. The K-Ras4B-PDE6δ stabilizer, ¹³¹I-C19, was highly selective and demonstrated a cytotoxic effect ten times greater than unlabeled I-C19. I-C19 prevented K-Ras4B activation and decreased its dependent signaling pathways. The in vivo administration of I-C19 (30 mg/kg) greatly reduced tumor growth in colorectal cancer. The biokinetic profile showed renal and hepatobiliary elimination, and the highest radiation absorbed dose was delivered to the tumor (52 Gy/74 MBq). The data support the idea that ¹³¹I-C19 is a novel K-Ras4B/PDE6δ stabilizer with two functionalities: as a K-Ras4B signaling inhibitor and as a compound with radiotherapeutic activity against colorectal tumors.

Keywords: I-C19; K-Ras4B; PDE6δ; colorectal cancer; pharmacokinetics.

Figure 1

(A) RMSD and (B) Rg values of the complexes between KRas4B^WT–PDE6δ or KRas4B^G13D–PDE6δ with C19 or I-C19. RMSD (left) values of KRas4B^WT–PDE6δ-C19 (black line) KRas4B^G13D–PDE6δ-C19 (red line), KRas4B^WT–PDE6δ-I-C19 (blue line), and KRas4B^G13D–PDE6δ-I-C19 (magenta line). Rg (right) values of KRas4B^WT–PDE6δ-C19 (black line), KRas4B^G13D–PDE6δ-C19 (red line), KRas4B^WT–PDE6δ-I-C19 (blue line) and KRas4B^G13D–PDE6δ-I-C19 (magenta line).

Figure 2

Most frequent conformations of C19 and I-C19 within the K-Ras4B–PDE6δ (cyan–yellow) complex during the MD simulations: (A) K-Ras4B–PDE6δ–C19; (B) K-Ras4B^G13D–PDE6δ-C19; (C) K-Ras4B–PDE6δ–I-C19; (D) K-Ras4B^G13D–PDE6δ–I-C19. Labels A, B and C denote residues from HVR2, PDEδ and K-Ras4B, respectively. The dotted lines represent hydrogen bonds.

Figure 3

Reversed-phase radio-HPLC chromatogram of the ¹³¹I-labeled C19 compound. General scheme of I-C19 structure labeled with ¹³¹I (top-right).

Figure 4

The colorectal cancer cell lines (A) HCT116; (B) LoVo; (C) SW620 treated with the indicated concentrations of I-C19 for 72 h (p < 0.05); (D) treatment with ¹³¹I-C19 at a concentration of 1.8, 6.8 and 8.8 µM in LoVo, SW620 and HCT116, respectively, significantly reduced the viability of the three colorectal cancer cell lines in comparison with unlabeled I-C19; (E) ¹³¹I-C19 uptake experiments. After incubation for 1, 3 and 5 h, ¹³¹I-C19 uptake by cancer cells significantly increased. n = 3; **** p < 0.0001.

Figure 5

(A) Representative images of the confocal microscope showing γ-H2AX foci in the nucleus (DAPI) of colorectal cancer cells treated with the vehicle and the IC₅₀ of I-C19 (a tenth of the IC₅₀ concentrations of each cell line) for 3 h; (B) relative γ-H2AX foci of cancer cells treated with I-C19, ¹³¹I-C19 and control cells. Data are presented as the mean ± SEM from three independent experiments. * p < 0.1; **** p < 0.0001.

Figure 6

(A) RAS-GTP pull-down assay of three colorectal cancer cell lines treated with the vehicle and the IC₅₀ of I-C19 for 24 h, and the total Ras and β-actin were the loading controls (cropping blots); (B) human phospho-kinase array of the total protein extract of the colorectal cancer HCT116 cell line treated with the IC₅₀ of I-C19 for 24 h. (C) human phospho-kinase array of the total protein extract of the colorectal cancer LoVo cell line treated with the IC₅₀ of I-C19 for 24 h. (D) human phospho-kinase array of the total protein extract of the colorectal cancer SW620 cell line treated with the IC₅₀ of I-C19 for 24 h. Pixel density—% of control.

Figure 7

(A) The effect of I-C19 treatment on tumor growth of AOM/DSS-treated mice, showing the percentage of colon adenocarcinoma among the mice. The mice were given C19 (30 mg/kg) or I-C19 (30 mg/kg) intraperitoneally every day for 12 days. (B) Body weight percentage of AOM/DSS-treated mice, measured every day during treatment. (C) Representative pictures of IHC staining for CEA and Ki67 in colorectal tissues. Data are presented as the mean ± SEM from independent experiments ** p < 0.01, *** p < 0.001.

Figure 8

(A) I-C19 was administered every day for 12 days at a concentration of 30 mg/kg. Tumor volume was measured daily and estimated according to the formula: V =(½ d)(D²); **** p < 0.001. (B) Representative images of tumor volume after dissection. (C) Body weight percentage for tumor-bearing nude mice measured every day during treatment. (D) Representative images of IHC staining for CEA, Ki67 and cleaved caspase-3 in the xenograft tumor tissues. (E) Graphs showing the percentage of neoplastic tissue positive to stain; * p < 0.1.

Figure 9

Biokinetic profile of (A) I-C19 [$q_h\left(t\right)$] and (B) ¹³¹I-C19 [$A_h\left(t\right)$ ] in Nu/Nu mice with LoVo xenograft tumors after intraperitoneal administration.

Figure 10

(A) Representative images of bone marrow cells from mice 24 h after a single injection of 15 mg/kg of 5-Fu and 30 mg/kg of C19 and I-C19. The arrows show the polychromatic erythrocytes (PCEs), micronuclei of polychromatic erythrocytes (MNPCEs) and normochromic erythrocytes (NCEs). (B) Values of micronuclei counts in the bone marrow cells. Data are presented as the mean ± SEM from four independent experiments **** p < 0.0001; n.s., non-significant.