PCW-1001, a Novel Pyrazole Derivative, Exerts Antitumor and Radio-Sensitizing Activities in Breast Cancer

Abstract

As pyrazole and its derivatives have a wide range of biological activities, including anticancer activity, the design of novel pyrazole derivatives has emerged as an important research field. This study describes a novel pyrazole derivative that exerts antitumor and radiosensitizing activities in breast cancer both in vitro and in vivo. We synthesized a novel pyrazole compound N,N-dimethyl-N'-(3-(1-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)phenyl)azanesulfonamide (PCW-1001) and showed that it inhibited several oncogenic properties of breast cancer both in vitro and in vivo. PCW-1001 induced apoptosis in several breast cancer cell lines. Transcriptome analysis of PCW-1001-treated cells showed that it regulates genes involved in the DNA damage response, suggesting its potential use in radiotherapy. Indeed, PCW-1001 enhanced the radiation sensitivity of breast cancer cells by modulating the expression of DNA damage response genes. Therefore, our data describe a novel pyrazole compound, PCW-1001, with antitumor and radiosensitizer activities in breast cancer.

Keywords: breast cancer; chemotherapy; combination therapy; pyrazole derivative; radio-sensitizer.

Figure 1

Synthesis of PCW-1001. (A) Reaction scheme to synthesize 11 different compounds containing pyrazole group. aReagents and conditions: (a) Cu(OAc)2, Cs2CO3, DMF, 110°C, 24 h. (b) Pd(OAc)2, K3PO4, sSPhos, dioxane, H2O, 80°C, 2 h. (c) Pyridine, 0°C to rt. 8 h (d) K2CO3, 80°C, 2 h. (e) Pd(PPh3)4, Na2CO3, dioxane, H2O, 80°C, 2 h. (f) KOAc, EtOH, room temperature, 2 h. (g) Pyridine, room temperature, 2 h. (B) The N,N-dimethylsulfonyl urea group exhibit charge–charge interaction, hydrogen bonding acceptor, and hydrogen bonding donor characteristics.

Figure 2

PCW-1001 inhibited several oncogenic properties of breast cancer cells. (A) The chemical structure of PCW-1001. (B) Breast cancer cell lines, including T47D, BT549, MDA-MB-231, MCF7, and MDA-MB-453, were treated with the indicated concentration of PCW-1001 for 72 h, and cell viability was analyzed. (C, D) The same cell lines were treated with 10 μM PCW-1001 for 14 days, and colony formation (C) and mammosphere formation (D) were analyzed. Upper panels are representative images and lower graphs are quantification data using ImageJ software or DIXI image solution. Scale bars = 100 µm. The data represent the results and are presented as mean ± standard deviation of three independent experiments. *P < 0.01.

Figure 3

PCW-1001 reduced tumor growth of breast cancer cells in vivo. Female BALB/c nude mice were subcutaneously injected with 5 × 10⁶ BT549 cells and treated with DMSO (Ctrl) or PCW-1001 (30 mg/kg) twice a week. (A–D) The tumor volume (A) and body weight (D) were measured periodically as indicated, and the tumor weights (B) and images (C) were obtained at the end of the experiment. (E) Cell lysates of the tumor were analyzed by immunoblotting with an anti-Ki67 antibody. β-actin was used as the loading control. The data represent the results and are presented as mean ± standard deviation of three independent experiments. N, not significant; *P < 0.05, **P < 0.01.

Figure 4

PCW-1001 induced apoptosis of breast cancer cell lines. Annexin-V assay was performed with BT549 cells treated with the indicated doses of PCW-1001 for 24 h (A) or 10 μM PCW-1001 for 48 or 72 h (B) to study apoptosis. (C–E) BT549, T47D, and MCF7 cells were treated with increasing doses of PCW-1001 for 24 h. Cell lysates were analyzed by immunoblotting with the indicated antibodies. β-actin was used as the loading control. The data represent the results and are presented as mean ± standard deviation of three independent experiments. N, not significant; *P < 0.01.

Figure 5

Gene expression analysis in PCW-1001-treated MCF7 cells. MCF7 was treated with DMSO (Ctrl) and 10 μM PCW-1001 for 24 h. The mRNA expression was analyzed using the nCounter^®PanCancer Pathway Panel that was designed to quantitate 770 genes. A heatmap of upregulated (red) and downregulated (green) genes (A) and lists of upregulated DNA damage response genes (B), upper panel) and down-regulated DNA damage repair genes (B), lower panel) were analyzed based on the database. Genes exhibited changes more than 1.5-fold. (C) Cell lysates were analyzed by qPCR. GAPDH was used as an internal control. The data represent the results and are presented as mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01.

Figure 6

PCW-1001 enhanced the radiation sensitivity of breast cancer cells. BT549, T47D, and MCF7 cells were treated with DMSO (Ctrl) and PCW-1001 (A; 2.5 μM, B, C; 10 μM) without or with 2 Gy radiation for 14 days, 1 h, or 24 h. (A) Colony formation was determined using the colony formation assay; representative images (upper panel) and the quantification (lower panel) of colony formation of each cell line treated with indicated conditions. (B) Immunofluorescent staining was performed to measure the level of γH2AX, and the intensities of γH2AX were quantified using the IN Cell Analyzer HCA System. (C) Cell lysates were analyzed by immunoblotting with the indicated antibodies. β-actin was used as the loading control. The data represent the results and are presented as mean ± standard deviation of three independent experiments. N, not significant; *P < 0.05; **P < 0.01.