Inhibitory effects of N-(4-hydrophenyl) retinamide on liver cancer and malignant melanoma cells

Abstract

Aim: To investigate the effect of N-(4-hydrophenyl) retinamide (4-HPR), the derivative of retinoic acid, on inhibition of migration, invasion, cell growth, and induction of apoptosis in hepatocellular carcinoma cells (HCCs) and malignant melanoma cells.

Methods: 4-HPR was chemically synthesized. Cellular migration and invasion were assayed by Borden chamber experiment. Cell growth was assayed by MTT chromometry. Apoptosis effect was measured using Hoechst 32258 staining and flow cytometry. Gene transfection was performed with lipofectamine.

Results: We observed that the migration of HCC and melanoma cells was significantly suppressed by 4-HPR and the migration cells were reduced to 58+/-5.03 (control 201+/-27.2, P<0.05, n = 4) in SMMC 7721-k3 HCC, and to 254+/-25.04 (control 302+/-30.1, P<0.05, n = 4) in melanoma cells after 6-h incubation with 4-HPR. The invasion through reconstituted basement membrane was also significantly reduced by 4-HPR treatment to 11.2+/-3.3 in SMMC 7721-k3 HCC (control 27+/-13.1), and to 24.3+/-3.2 in melanoma cells (control 67.5+/-10.1, P<0.05, n = 3). Cell growth, especially in melanoma cells, was also significantly inhibited. Furthermore, 3 micromol/L of 4-HPR induced apoptosis in B16 melanoma cells (37.11+/-0.94%) more significantly than all-trans retinoic acid (P<0.05), but it failed to induce apoptosis in SMMC 7721-k3 HCC. The mechanism for 4-HPR-induced apoptosis was not clear, but we observed that 4-HPR could regulate p27(kip1), and overexpression of cerebroside sulfotransferase (CST) diminished the apoptosis induced by 4-HPR in melanoma cells.

Conclusion: 4-HPR is a potent inhibitor of HCC migration and inducer of melanoma cell apoptosis. CST and p27(kip1) expression might be associated with 4-HPR-induced apoptosis.

Figure 1

Inhibition migration of hepatocellular carcinoma and melanoma cells by 4-HPR treatment within a short period. SMMC 7721-k3 and B16 melanoma cells were treated with 4-HPR for 6, 18, and 24 h, respectively. After treatment, the cells were assayed for migration in the Transwell.

Figure 2

Effect of 3 mmol/L 4-HPR on cell invasion. SMMC 7721-k3 and B16 melanoma cells were treated with 4-HPR for 6 h. The cells penetrating matrigel membrane and migrating to the bottom of the filter were counted and data were expressed as an average from six independent assays.

Figure 3

Growth inhibition of SMMC 7721-k3 cells by ATRA and 4-HPR. SMMC 7721-k3 cells were cultured in the 96-well cluster plate and incubated with 10 mmol/L ATRA, 3 mmol/L 4-HPR, or ethanol in the control.

Figure 4

Growth inhibition of B16 melanoma cells by ATRA and 4-HPR. B16 melanoma cells were cultured in the 96-well cluster plate and incubated with 10 mmol/L ATRA, 3 mmol/L 4-HPR, or ethanol in the control. The data were achieved by an average from eight independent groups.

Figure 5

Regulation of cell cycles by 4-HPR. SMMC 7721-k3 cells and B16 melanoma cells were treated with 4-HPR for 24 h, respectively. Data were expressed as an average from three independent assays.

Figure 6

Western blot analysis of effects of 4-HPR on p27kip1 and p21waf1 expression. SMMC 7721-k3 cells were treated with 10 and 3 mmol/L 4-HPR for 24 h, respectively.

Figure 7

Comparison of apoptosis peak between CST- and vector-transfected cells. Control cells either of vector- or CST-transfected could not show any apoptosis (A). After being treated with 4-HPR, the vector-transfected cells produced a large apoptosis peak (B), while in CST-transfected cells only a small apoptosis peak could be seen (C).