Novel Thymoquinone Derivative TQFL28 Inhibits Triple-Negative Breast Cancer (TNBC) Invasiveness In Vitro and In Vivo

Abstract

Although thymoquinone (TQ) has been reported as an anti-tumor small molecule well investigated in numerous tumors. In this study, we designed and synthesized a novel TQ derivative, TQFL28, with a molecular formula of C₂₀H₂₃NO₂. TQFL28 showed stronger cytotoxicity or anti-proliferative activities against triple-negative breast cancer (TNBC) cell lines (BT549, MDA-MB-231, or 4T1) than TQ but is lower in the normal mammary epithelial cells, MCF10A. TQFL28 exhibited lower IC₅₀ values toward BT549 (38.78 ± 1.589) and MDA-MB-231 (39.63 ± 1.598) cells compared to TQ, indicating its efficacy for TNBC cytotoxicity. TQFL28 inhibited the growth, migration, and invasiveness of TNBC cells of 4T1 and BT549 in vitro and tumor progression and metastasis in a 4T1 allograft animal model in vivo. Moreover, TQFL28 presents lower toxicity than TQ in mice, showing a 7-day half-lethal dose (LD₅₀) of 59.43 mg/kg (41.6-83.6, 95% confidence interval). Altogether, our study obtained. In addition, TQFL28 induced a significant reduction in tumor volumes in the mouse model in comparison to the vehicle group. TQFL28, a novel small molecule, has a superior inhibitory effect and lower toxicity on TNBC both in vitro and in vivo. Thus, TQFL28 might have potential as a therapeutic small molecule for breast cancer, especially in TNBC.

Keywords: TQFL28; anti-cancer; derivative; invasiveness; thymoquinone; triple-negative breast cancer.

Figure 1

The synthetic route and structure for TQFL28. NTQ is 3-amino-5-isopropyl-2-methylcyclohexa-2,5-diene-1,4-dione [30]. TQFL28 is (E)-5-isopropyl-3-((4-isopropylbenzylidene)amino)-2-methylcyclohexa-2,5-diene-1,4-dione. The molecular formula for TQFL28 is C₂₀H₂₃NO₂. We developed the TQFL28 small molecule by adding a side chain at C-6 of TQ, since synthesized derivatives showed that the compounds with unsaturated side chains confer higher activities than those with equally long saturated chains. NaN₃ is sodium azide.

Figure 2

Effects of TQFL28 and TQ on the viability of different breast cancer cells and normal breast epithelial cells. (A,B) CCK8 assays show the effect of TQFL28 and TQ at 24 h in BT549 (A) and MDA-MB-231 (B), respectively. (C) The effect of TQFL28 and TQ on 24 h in MCF10A (normal breast epithelial cell line). (D–F) The apoptotic effect of TQFL28 in 4T1 at the indicated TQFL28 concentrations. A total of 0 µM indicates no TQFL28 treatments. The results are presented as the mean ± SD (n = 3).

Figure 3

TQFL28 suppresses breast cancer cell growth, migration, and invasion. (A–C) TQFL28 suppresses breast cancer cell growth (A), migration (B), and invasion (C) in the indicated concentrations of 4T1 cells. (D–F) TQFL28 suppresses breast cancer cell growth (D), migration (E), and invasion (F) in the indicated concentrations of BT549 cells. The Y-axis indicates the cell index (cell numbers), while the X-axis indicates the treated time (h).

Figure 4

Effect of TQFL28 on breast cancer tumor growth in mice’s allograft. (A) TQFL28 suppresses tumor growth with the tumor size in a dose-dependent manner. (B) TQFL28 suppresses tumor growth measured with the tumor weights in a dose-dependent manner. (C) The quantitative data for (B). Representative images for in situ tumors without (D) or with (E,F) TQFL28 treatments (0, 3, or 7.5 mg/kg, respectively). “*” indicates p < 0.05 and “**” indicates p < 0.01. Vehicle: the vehicle-treated group. Scale bar = 5 μM.

Figure 5

TQFL28 treatment suppresses breast cancer metastasis to the lungs. (A) The size of metastasis colonies on the lungs was measured in TQFL28 treatment with 0 mg/kg, 3 mg/kg, or 7.5 mg/kg, respectively. (B) The number of metastasis nodules on the lungs after TQFL28 treatment. (C) The tumor length on the lungs after TQFL28 treatment (percentages to control). (D) Representative images for metastatic tumors on the lungs without TQFL28 treatment. (E) Representative images for lung metastatic tumors by 3 mg/kg TQFL28 treatments. (F) Representative images for lung metastatic tumors by 7.5 mg/kg TQFL28 treatments. “*” indicates p < 0.05 and “****” indicates p < 0.0001. Scale bar = 5 μM.