RGFP966 Suppresses Tumor Growth and Migration Through Inhibition of EGFR Expression in Hepatocellular Carcinoma Cells in vitro

Abstract

Purpose: Histone deacetylase 3 (HDAC3) has been suggested to play a role in hepatocellular carcinoma (HCC). In the present report, we aimed to identify the effects of RGFP966, a specific HDAC3 inhibitor, on the cell proliferation and migration of HCC cell lines.

Methods: Human HCC cell lines, which were identified using short tandem repeat (STR) DNA profiling analysis, were used in this report. Cell proliferation assay was used to identify the growth viability of cells. Wound healing and transwell assay were used to identify the migration ability of cells. Further, a human phospho-receptor tyrosine kinases array kit was used to screen out RGFP966 effects on key receptor tyrosine kinases. Then, the mRNA expression was quantified by real-time PCR, and protein expression was identified by Western blot immunoassay.

Results: We found that RGFP966 inhibited both proliferation and migration of HCC cells. Further, RGFP966 represses the expression and phosphorylation levels of epidermal growth factor receptor (EGFR) in HCC cells. Moreover, HDAC3 is involved in the inhibition of EGFR by RGFP966. Overall, we elucidated an inhibitive function of RGFP966 in HCC progression.

Conclusion: RGFP966 inhibits EGFR signaling pathway and suppresses proliferation and migration of HCC cells.

Keywords: EGFR; HDAC3; RGFP966; hepatocellular carcinoma.

Figure 1

Growth repression induced by RGFP966 in HCC cells. (A–C), PLC/PRL/5, Huh7 and HepG2 cells were treated with indicated doses of RGFP966, or vehicle. 48 hrs later, relative cell numbers were determined using MTS assay by absorbance at 492 nm. Data are represented as mean ± SD from three independent experiments. P value refers to two-sided t test. (D–F), PLC/PRL/5, Huh7 and HepG2 cells were treated with RGFP966 (10 or 25μM) or vehicle. Relative cell numbers were determined at indicated times using MTS assay by absorbance at 492 nm and normalized by 0 hr group. Data are represented as mean ± SD from three independent experiments. P value refers to two-sided t test.

Figure 2

RGFP966 suppresses cell migration of HCC cells. (A) and (B), 5x10 Huh7 and PLC/PRL/5 cells were plated into transwell chamber with treatment of RGFP966 (RGFP,10μM) or vehicle. After 40 hrs, the invaded cells were stained, and representative images were photographed. (C) and (D), After a linear wound was generated, Huh7 and PLC/PRL/5 cells were treated with RGFP966 (RGFP, 10μM) or vehicle. After 40 hrs, representative images were photographed.

Figure 3

RGFP966 downregulates the expression and phosphorylation levels of EGFR in HCC cells. (A), Huh7 cells were treated with or without RGFP966 (RGFP, 10μM). And Human Phospho-RTK array was used to detect the effect of RGFP966 on relative phosphorylation of 49 different RTKs. Representative images were shown. (B) and (C) after treatment with indicated concentrations of RGFP966 (RGFP) for 48 hrs, proteins from Huh7 (B) and PLC/PRL/5 (C) cells were harvested, and Western Blot analysis was performed with the indicated antibodies. GAPDH was used as internal control.

Figure 4

Inhibition of EGFR by RGFP966 is associated with HDAC3. (A), Huh7 cells were treated with indicated doses of RGFP966 (RGFP), or vehicle. After 40 hrs, cells were harvested, and total RNA was extracted. Relative expression of EGFR mRNA was determined by real-time PCR. GAPDH was used as internal control. Data are represented as mean ± SD from three independent experiments. P value refers to two-sided t test. (B), Huh7 cells were separately transfected with empty vector and expression vector for HA-tagged HDAC3. After 40 hrs, cells were harvested, and total RNA was extracted. Relative expression of EGFR mRNA was determined by real-time PCR. GAPDH was used as internal control. Data are represented as mean ± SD from three independent experiments. P value refers to two-sided t test. (C), Huh7 cells were separately transfected with empty vector and different doses of HA-tagged HDAC3 expression plasmids. After 48 hrs, cells were harvested, and Western Blot analysis was performed with the indicated antibodies. GAPDH was used as internal control. (D), Huh7 cells were separately transfected with empty vector and expression vector for HA-tagged HDAC3. After 24 hrs, 2.5 x10⁴ cells were plated into a transwell chamber. After 40 hrs, the invaded cells were stained, and representative images were photographed. (E), Huh7 cells were treated with indicated doses of RGFP966 (RGFP), or vehicle. After 48 hrs, cells were harvested and Western Blot analysis was performed with the indicated antibodies. GAPDH was used as internal control. (F), Huh7 cells were separately transfected with empty vector and expression plasmids for HA-HDAC3, and then were treated with RGFP966 (RGFP, 10μM) and vehicle for 48hrs. Then, cells were harvested, and Western Blot analysis was performed with the indicated antibodies. GAPDH was used as internal control.