A diaryl urea derivative, SMCl inhibits cell proliferation through the RAS/RAF/MEK/ERK pathway in hepatocellular carcinoma

Abstract

Introduction: Hepatocellular carcinoma (HCC) ranks among the three most prevalent cancer-related diseases in terms of incidence. Hence, exploring drugs for HCC therapy is of great significance. Compounds with a diaryl urea structure have been reported to exhibit a broad range of biological activities, including anticancer activity. This study focuses on the specific diaryl urea derivative 4-(4-(3-(2-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)-N-methylpicolinamide (SMCl), with particular emphasis on investigating its therapeutic effects against hepatocellular carcinoma (HCC) and elucidating the underlying molecular mechanisms.

Methods: In vitro anti-cancer effects of SMCl were evaluated in HCC cell lines using MTS, colony formation, and wound healing assays. Western blot analyzed RAS/RAF/MEK/ERK pathway modulation. In vivo efficacy was assessed using a xenograft model.

Results: The MTS and colony formation assays demonstrated that SMCl significantly decreased the viability of HCC cells. Western blot analysis demonstrated that SMCl effectively suppressed hepatocellular carcinoma proliferation by markedly inhibiting the RAS/RAF/MEK/ERK signaling pathway, with this inhibitory effect exhibiting both time- and concentration-dependent characteristics. SMCl also demonstrated significant therapeutic efficacy in the xenograft tumor model, achieving a tumor inhibition rate of 72.37%. Notably, it showed no significant impact on spleen weight or body weight in mice, indicating low toxicity to normal tissues.

Conclusion: This study first elucidates the effects of SMCl on HCC cells and its impact on the RAS/RAF/MEK/ERK signaling pathway, providing a potential active compound for the clinical treatment of liver cancer.

Keywords: RAS/RAF/MEK/ERK; bioactive compound; cell proliferation; diaryl urea derivative; hepatocellular carcinoma.

FIGURE 1

Effects of SMCl on the cell viability of HCC cells. (A,B) Chemical structure of SMCl and Sorafenib, (C–J) Effects of different concentrations of SMCl and Sorafenib on the proliferative ability of liver cancer cells at 72 h.

FIGURE 2

SMCl suppresses cell migration and colony formation. (A,B) Cells were treated with 0, 2.5, 5, and 10 μM SMCl for the indicated time. (C,D) Relative colony number at different concentration of SMCl. (E,F,I) Wound healing assay was performed. Cells were treated with 10 μM SMCl at 0, 24, 36, and 48 h. Scale bar, 100 μm. Experiments were independently conducted in triplicate. (G,H,J) The area of non-migrant cells was quantified using ImageJ software. Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, and ***p < 0.001 vs. the control group).

FIGURE 3

Investigated the impact of SMCl on the Ras/Raf/MAPK pathways in HCC cell lines. (A) Hep3B cells were divided into a control group and an SMCl treatment group (10 μM). After 48 h of treatment, fluorescence microscopy showed a marked reduction in the number of EdU-positive cells in the SMCl-treated group compared to the control group. (B) Effects of 0, 2.5, 5, and 10 μM SMCl treatment for 48 h on protein levels of Ras, Raf, Erk, and p21 in both Hep3B and PLC/PRF/5 cell lines. (C) Time-dependent effects of 10 μM SMCl treatment at 0, 24, 36, and 48 h on protein levels of Ras, Raf, Erk, and p21 in Hep3B and PLC/PRF/5 cells.

FIGURE 4

SMCl inhibits tumor progression in the SMCC7721 xenograft model. (A) Nude mouse bearing xenografted tumors were randomly divided into two groups: one as the control group treated daily with DMSO, and the other treated with SMCl 50 mg/kg/day for 12 days. Dissected tumors were obtained from mice in both the control and SMCl groups. (B) Tumor weight of subcutaneous tumors from the control and SMCL groups. (C) Comparison of subcutaneous tumor volumes from the control and SMCL groups (n = 5). (D) Comparison of body weight of nude mice from the control and SMCL groups. (E,F) Protein levels of pRaf, pErk, and Ras in subcutaneous tumors from the DMSO and SMCL groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5

SMCl exerts anti-HCC effects by targeting the RAS/RAF/MEK/ERK pathway.