4-Amino-2-trifluoromethyl-phenyl retinate inhibits proliferation, invasion, and migration of breast cancer cells by independently regulating CRABP2 and FABP5

Abstract

Background: 4-Amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel retinoid derivative, inhibits proliferation and induces differentiation in many cancer cells. In this study, the inhibitory effects of ATPR on the proliferation, invasion, and migration of breast cancer (BC) cells, and the relationship between ATPR and the expression of the intracellular lipid-binding proteins CRABP2 and FABP5 were investigated.

Methods: CRABP2 and FABP5 expression was evaluated in infiltrating breast-infiltrating ductal carcinoma(BIDC) and benign breast fibroma (BBF) by immunohistochemistry and in MCF-7, MDA-MB-231, MDA-MB-435, and MDA-MB-453 cells by immunofluorescence. The inhibition of proliferation by ATPR in these cells was detected by MTT. After downregulation and upregulation of CRABP2 and FABP5 in MCF-7 or MDA-MB-231 cells using siRNA and plasmids, the effect of ATPR on proliferation was detected by MTT and real-time cell analysis, and the effects of ATPR on the invasion and migration of MDA-MB-231 cells were detected using a Boyden chamber assay and a wound healing assay.

Results: CRABP2 expression was moderately or strongly positive in BIDC and BBF. FABP5 expression was also moderately or strongly positive in BIDC, but weakly positive or negative in BBF. CRABP2 and FABP5 were highly expressed in MCF-7 cells, moderately expressed in MDA-MB-453 cells, and weakly expressed in MDA-MB-435 and MDA-MB-231 cells. ATPR inhibited proliferation more strongly in MCF-7 cells than in other cells. The inhibition of proliferation by ATPR depended on an increase in CRABP2, but not FABP5 expression. A decrease in FABP5 could inhibit the invasion and migration of BC cells.

Conclusion: These findings indicate that ATPR might inhibit proliferation by upregulating CRABP2, and inhibit invasion and migration by downregulating FABP5 in BC cells. These findings may facilitate the use of differentiation therapy in BC.

Keywords: ATPR; CRABP2; FABP5; breast cancer; invasion; migration; proliferation.

Figure 1

Structure of 4-amino-2-trifluoromethyl-phenyl retinate (ATPR).

Figure 2

Expression of CRABP2 and FABP5 in breast cancer tissues and cells. Notes: (A) Expression of CRABP2 and FABP5 in breast cancer and breast benign fibroma was detected by immunohistochemistry. The staining patterns were scored as negative (−) if no immunolabeling was observed, weakly positive (+) if the labeling was faint, moderately positive (++) if the labeling was stronger, and strongly positive (+++) if the labeling was distinctly stronger than (++). Representative images are shown at 100× objective. (B) Expression of CRABP2 and FABP5 in different breast cancer cells was detected by immunofluorescence. Representative images are shown at 200× objective.

Figure 3

Rate of inhibition of breast cancer cells treated with various concentrations of 4-amino-2-trifluoromethyl-phenyl retinate (ATPR) or all-trans retinoic acid (ATRA) for 24, 48, and 72 h ($\overline{x}\pm s$, n = 3). MCF-7, MDA-MB-231, MDA-MB-435, and MDA-MB-453 cells.

Figure 4

Effects of 4-amino-2-trifluoromethyl-phenyl retinate (ATPR) on proliferation of MCF-7 cells after silencing of CRABP2. Notes: (A) Immunofluorescence staining on MCF-7 cells confirms significant reduction of CRABP2 expression in the CRABP2-siRNA group. Representative images obtained with a 200× objective. (B) An MTT assay was performed to evaluate transfected MCF-7 cells that were untreated or treated with 1 × 10⁻⁶ mol/L ATPR for 24, 48, and 72 h. *P < 0.05, ***P < 0.001. For 48 h, ATPR(−) siRNA(−) vs ATPR(+) siRNA (one-way ANOVA, P < 0.001), ATPR(+) negative siRNA(+) vs ATPR(+) CRABP2 siRNA(+) (one-way ANOVA, P = 0.048). For 72 h, ATPR(−) siRNA(−) vs ATPR(+) siRNA (one-way ANOVA, P < 0.001), ATPR(+) negative siRNA(+) vs ATPR(+) CRABP2 siRNA(+) (one-way ANOVA, P = 0.034).

Figure 5

Effects of 4-amino-2-trifluoromethyl-phenyl retinate (ATPR) on the proliferation of MDA-MB-231 cells after overexpression of CRABP2. Notes: (A) Immunofluorescence staining on MDA-MB-231 cells confirms significantly increased CRABP2 expression in the MDA-MB-231 (CP+) group. Representative images obtained using a 200× objective. (B) A real-time cell analysis was performed on transfected MDA-MB-231 cells that were untreated or treated with 1, 10, and 100 µM ATPR. CP−: cells untreated with CRABP2 transfection; CP+: cells treated with CRABP2 transfection.

Figure 6

Effects of 4-amino-2-trifluoromethyl-phenyl retinate (ATPR) on proliferation of MCF-7 cells after silencing of the FABP5 gene. Notes: (A) Immunofluorescence staining of MCF-7 cells confirms a significant reduction in FABP5 expression in the FABP5-siRNA group. Representative images were obtained using a 200× objective. (B) An MTT assay was performed on transfected MCF-7 cells that were untreated or treated with 1 × 10⁻⁶ mol/L ATPR for 24, 48, and 72 h. *P < 0.05. For 48 h, ATPR(−) siRNA(−) vs ATPR(+) siRNA(−) (one-way ANOVA, P = 0.012), ATPR(−) FABP5 siRNA(+) vs ATPR(+) FABP5 siRNA(+) (one-way ANOVA, P = 0.167), ATPR(−) siRNA(−) vs ATPR(−) FABP5 siRNA(+) (one-way ANOVA, P = 0.258). For 72 h, ATPR(−) siRNA(−) vs ATPR(+) siRNA(−) (one-way ANOVA, P = 0.018), ATPR(−) siRNA(−) vs ATPR(−) FABP5 siRNA(+) (one-way ANOVA, P = 0.019), ATPR(+) siRNA(−) vs ATPR(+) FABP5 siRNA(+) (one-way ANOVA, P = 0.943), ATPR(+) siRNA(−) and ATPR(+) FABP5 siRNA(+) (one-way ANOVA, P = 0.998). “NS” is not significant.

Figure 7

Effects of 4-amino-2-trifluoromethyl-phenyl retinate (ATPR) on the proliferation of MDA-MB-231 cells after overexpression of FABP5. Notes: (A) Immunofluorescence staining of MDA-MB-231 cells confirms significant increase in FABP5 expression in MDA-MB-231 (FL+) group. Representative images shown at 200× objective. (B) A real-time cell analysis performed on transfected MDA-MB-231 cells that were untreated or treated with 0.5 or 50 µM ATPR. FL−: cells untreated with FABP5 transfection; FL+: cells treated with FABP5 transfection.

Figure 8

Effects of 4-amino-2-trifluoromethyl-phenyl retinate (ATPR) on the invasion and migration of MDA-MB-231 cells after silencing and overexpression of FABP5. Notes: (A) Boyden chamber assay. MDA-MB-231 cells – with FABP5 silenced or overexpressed – were plated in the invasion chamber. After 20 h, cells remaining in the upper chamber were removed and cells that invaded through the Matrigel were fixed and stained using 0.5% crystal violet in methanol, ***P < 0.001 vs FL−/siRNA− group. (B) Wound healing assay. MDA-MB-231 cells – with FABP5 silenced or overexpressed – were subjected to a wound assay and monitored for 48 h. FL−: cells untreated with FABP5 transfection; FL+: cells treated with FABP5 transfection. (C) Immunofluorescence staining of FABP5 expression after treatment with ATPR (8 µmol/L) for 48 h (200×). One-way ANOVA, ***P < 0.001 vs the non-ATPR intervention group.