A Novel Paclitaxel Derivative for Triple-Negative Breast Cancer Chemotherapy

Abstract

Paclitaxel-triethylenetetramine hexaacetic acid conjugate (PTX-TTHA), a novel semi-synthetic taxane, is designed to improve the water solubility and cosolvent toxicity of paclitaxel in several aminopolycarboxylic acid groups. In this study, the in vitro and in vivo antitumor effects and mechanisms of PTX-TTHA against triple-negative breast cancer (TNBC) and its intravenous toxicity were evaluated. Results showed the water solubility of PTX-TTHA was greater than 5 mg/mL, which was about 7140-fold higher than that of paclitaxel (<0.7 µg/mL). PTX-TTHA (10-10⁵ nmol/L) could significantly inhibit breast cancer proliferation and induce apoptosis by stabilizing microtubules and arresting the cell cycle in the G2/M phase in vitro, with its therapeutic effect and mechanism similar to paclitaxel. However, when the MDA-MB-231 cell-derived xenograft (CDX) tumor model received PTX-TTHA (13.73 mg/kg) treatment once every 3 days for 21 days, the tumor inhibition rate was up to 77.32%. Furthermore, PTX-TTHA could inhibit tumor proliferation by downregulating Ki-67, and induce apoptosis by increasing pro-apoptotic proteins (Bax, cleaved caspase-3) and TdT-mediated dUTP nick end labeling (TUNEL) positive apoptotic cells, and reducing anti-apoptotic protein (Bcl-2). Moreover, PTX-TTHA demonstrated no sign of acute toxicity on vital organs, hematological, and biochemical parameters at the limit dose (138.6 mg/kg, i.v.). Our study indicated that PTX-TTHA showed better water solubility than paclitaxel, as well as comparable in vitro and in vivo antitumor activity in TNBC models. In addition, the antitumor mechanism of PTX-TTHA was related to microtubule regulation and apoptosis signaling pathway activation.

Keywords: antitumor; apoptosis; microtubule stabilization; proliferation; triple-negative breast cancer.

Figure 1

Chemical structure and characterization of PTX-TTHA. Chemical structure of (A) PTX and (B) PTX-TTHA. (C) Purity of PTX-TTHA analyzed by HPLC (Waters SunFire^TM C18 column 4.6 × 250 mm), eluted by A/B = 95:5 (A 0.01 M KH₂PO₄ aqueous solution: acetonitrile = 54:46, v/v; B 0.01 M KH₂PO₄ aqueous solution, phosphoric acid adjusts the pH to about 2.0) at a flow rate of 1 mL/min, and absorbance recorded at 227 nm. (D) Images of the water solubility between PTX-TTHA, Taxol, and paclitaxel at 5 mg/mL.

Figure 2

PTX-TTHA inhibited breast cancer cell proliferation and induced apoptosis in a dose-dependent manner. The cell viabilities of (A) MDA-MB-231, (B) MCF-7, (C) 4T1, and (D) MCF 10A after 48 h treatment with PTX-TTHA, compared to PTX. The concentration of PTX and PTX-TTHA ranged from 0.01 nM to 100 μM. (E) After being treated with PTX (0.1 nM) or PTX-TTHA (0, 0.1, 1 nM) for 48 h, MDA-MB-231 cells were stained with Annexin V-FITC and propidium iodide, and then analyzed by flow cytometry. The percentage of surviving cells is shown in the lower left Q4 quadrant, and the percentages of early-stage and late-stage apoptotic cells are shown in the lower right Q3 and upper right Q2 quadrants, respectively. (F) The apoptosis induced by PTX or PTX-TTHA was quantified. (G) Activation of caspase-3 activity in MDA-MB-231 cells after 48 h of PTX (0.1 nM) or PTX-TTHA (0, 0.1, 1 nM) treatment. The results are presented as the mean ± SD of three independent experiments. Control: 0 nM PTX-TTHA treated MDA-MB-231 cells. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. the control group.

Figure 3

PTX-TTHA-induced morphological changes of apoptosis in MDA-MB-231 cells. Representative transmission electron micrographs of (A) 0 nM PTX-TTHA, (B) 0.1 nM PTX, (C) 0.1 nM PTX-TTHA treated MDA-MB-231 cells. (D–F) Enlargement of the panels in 5A, 5B, and 5C. (Magnification: ×4800 and ×18,500, scale bars: 2 μm (upper) and 500 nm (lower)). Control: 0 nM PTX-TTHA treated MDA-MB-231 cells.

Figure 4

PTX-TTHA exerted antitumor activity by stabilizing microtubules and arresting cells in the G2/M phase. (A) Microtubule stabilization images of PTX-TTHA in MDA-MB-231 cells. Microtubules were observed using the α-tubulin antibody, followed by the Alexa Fluor 488 conjugated secondary antibody (green). The nuclei were stained with DAPI (blue) (magnification: ×630, scale bars: 20 μm). (B) Semi-quantitative analysis of microtubule fluorescence intensity using Image-J software. The value of fluorescence intensity in the control group was normalized to 1. (C) Cell cycle distribution of MDA-MB-231 cells after treatment with PTX (0.1 nM) or PTX-TTHA (0, 0.1, 1 nM) for 48 h. The blue peak represents cells in the G0/G1 phase, the green area represents cells in the S phase, and the pink peak represents cells in the G2/M phase. (D) Quantification of the percentage of cell cycle distribution. The bars indicate mean ± SD (n = 3). Control: 0 nM PTX-TTHA treated MDA-MB-231 cells. * p < 0.05, *** p < 0.001, vs. control group.

Figure 5

PTX-TTHA reduced the tumor size in vivo. MDA-MB-231 breast tumor-bearing mice treated with saline (Ctrl), 10 mg/kg PTX (PTX), and 6.87 (LPTX-TTHA), 10.30 (MPTX-TTHA), 13.73 (HPTX-TTHA) mg/kg PTX-TTHA, which were equimolar with 5, 7.5, and 10 mg/kg PTX, respectively. (A) Weight change during the 3-week treatment course. (B) Weight of separated tumors. (C) Time course of mean tumor volume during treatment. (D) Enlargement of tumor volume change in 6C. Each point represents the mean ± SD (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Antitumor efficacy of PTX-TTHA in the MDA-MB-231 xenograft breast tumor model. (A) Western blot analysis was performed on Bcl2, Bax, cleaved caspase-3, and β-actin using tumor tissues. (B–D) The bar graphs represent the densitometric analysis of the protein expression levels using image J software from three different experiments. The value of β-actin expression level was normalized to 1. (E) The separated tumor tissues were used for H&E staining (magnification: ×400, scale bars: 50 μm), immunohistochemistry (IHC) analysis of Ki-67 (magnification: ×200, scale bars: 100 μm), and TUNEL assay (magnification: ×400, scale bars: 50 μm). (F) Semi-quantitative analysis of the percentage of Ki-67 positive area over total area. (G) Semi-quantitative analysis of TUNEL-positive cells. For H&E staining, tumor cells with active proliferations are shown in the Ctrl group, such as chromosomes gathering in the equatorial plate (circle sign) and stretching back to the poles of the cell (square sign). Apoptotic cells (arrow sign) are observed in PTX and PTX-TTHA treatment groups. The results are expressed as mean ± SD (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 7

The acute toxicity of PTX-TTHA in vivo. Mice were treated with 138.6 mg/kg PTX-TTHA, 20 mg/kg CrEL paclitaxel, Taxol (PTX), and 0.9% normal saline and then observed for 14 days. (A) Time course of mean body weight change after administration. (B,C) Blood biochemical and hematological tests on Day 3 and Day 14. (D) Organ-body indices. (E) Photographs of organs stained with H&E. (All magnification: ×200, scale bars: 100 μm). (Circle sign) Histopathological changes in the liver and kidney tissue of the PTX group. Each point represents the mean ± SD (n = 10). * p < 0.05, ** p < 0.01, *** p < 0.001.