Colchicine-Binding Site Agent CH-2-77 as a Potent Tubulin Inhibitor Suppressing Triple-Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) is a highly aggressive type of breast cancer. Unlike other subtypes of breast cancer, TNBC lacks hormone and growth factor receptor targets. Colchicine-binding site inhibitors (CBSI) targeting tubulin have been recognized as attractive agents for cancer therapy, but there are no CBSI drugs currently FDA approved. CH-2-77 has been reported to have potent antiproliferative activity against a panel of cancer cells in vitro and efficacious antitumor effects on melanoma xenografts, yet, its anticancer activity specifically against TNBC is unknown. Herein, we demonstrate that CH-2-77 inhibits the proliferation of both paclitaxel-sensitive and paclitaxel-resistant TNBC cells with an average IC50 of 3 nmol/L. CH-2-77 also efficiently disrupts the microtubule assembly, inhibits the migration and invasion of TNBC cells, and induces G2-M cell-cycle arrest. The increased number of apoptotic cells and the pattern of expression of apoptosis-related proteins in treated MDA-MB-231 cells suggest that CH-2-77 induces cell apoptosis through the intrinsic apoptotic pathway. In vivo, CH-2-77 shows acceptable overall pharmacokinetics and strongly suppresses the growth of orthotopic MDA-MB-231 xenografts without gross cumulative toxicities when administered 5 times a week. The in vivo efficacy of CH-2-77 (20 mg/kg) is comparable with that of CA4P (28 mg/kg), a CBSI that went through clinical trials. Importantly, CH-2-77 prevents lung metastasis originating from the mammary fat pad in a dose-dependent manner. Our data demonstrate that CH-2-77 is a promising new generation of tubulin inhibitors that inhibit the growth and metastasis of TNBC, and it is worthy of further development as an anticancer agent.

Figure 1.

CH-2-77 impairs the colony formation and microtubule organization and stability of TNBC cells. (A) Chemical structure of CH-2-77. (B) Bar graphs show the anti-colony formation effects of CH-2-77 on MDA-MB-231 or MDA-MB-468 cells compared to colchicine and paclitaxel in the low nanomolar range (1 to 4 nM). The percentage (%) of colony area in each treatment group was quantified by comparing to the colony area observed in the control group. (C) MDA-MB-231 cells treated with 2.5 nM or 5 nM of colchicine, paclitaxel, or CH-2-77 were imaged using a Keyence microscope (×20 magnification). Inserts shown in the bottom right corner show tubulin for mitotic cells (×40 magnification). Cells were stained for α-tubulin (red) and the nucleus (blue). (D) The expression of α-tubulin and acylated-α-tubulin in CH-2-77-treated MDA-MB-231 cells (24 h) was determined by western blot. GAPDH is the loading control. (E-F) Immunocytochemistry and western blot experiments were repeated in MDA-MB-468 cells with similar conditions as in C and D.

Figure 2.

CH-2-77 inhibits cell migration and invasion of TNBC cells. (A) The wound healing assay showed the effects of colchicine, paclitaxel, and CH-2-77 to prevent wound closure in MDA-MB-231 cells or MDA-MB-468 cells at a concentration of 4 nM. Representative images were captured at indicated time points after drug treatment for both cell lines. % wound area of each treatment group was compared to their counterparts at the start point (0 h). P values were determined relative to the control group. (B-C) A Transwell chemotaxis migration using Transwell noncoated inserts (B) or invasion assay using Transwell chambers coated with Matrigel (C) was performed using TNBC cells treated with 4 nM of CH-2-77. Representative cell images were acquired from the lower wells. The migration or invasion potential of CH-2-77-treated TNBC cells was calculated as the percentage of migrated or invaded cells relative to the cells in the control group (set to 100%).

Figure 3.

CH-2-77 induces cell cycle arrest and cell apoptosis in MDA-MB-231 and MDA-MB-468 cells. (A) Effects of increasing concentrations of CH-2-77 (2.5 nM, 5 nM and 10 nM), or 10 nM of colchicine (COL) or 10 nM of paclitaxel (PTX) on TNBC cell cycle distribution after 24 h treatment as detected by phospho-histone H3 (Ser10)/PI co-staining. (B) Annexin-V/PI co-staining evaluated the extent of apoptosis in TNBC cells with the same drug treatments as in A. (C) Expression of cleaved-PARP was compared in both TNBC cell lines by western blot after treatment as in A. GAPDH was used as a loading control. (D) Western blot analysis was repeated after 24 h of exposure to CH-2-77 treatment to compare levels of full-length PARP and the cleaved active PARP counterpart (cleaved-PARP). β-actin was used as a loading control. (E) Expression of a panel of apoptosis-related proteins, cleaved-caspase-9, cleaved-caspase-3, Bax, and Bcl-2, were compared in MDA-MB-231 cells after CH-2-77 treatments for 24 h. Either GAPDH or β-actin was used as a loading control.

Figure 4.

Plasma concentration-time profile (geomean ± 95%CI) of CH-2-77 in rats (n=5) after intravenous administration of 5 mg/kg.

Figure 5.

CH-2-77 inhibits primary tumor growth and lung metastasis in an orthotopic MDA-MB-231 TNBC xenograft model in a dose-dependent manner. Mean tumor volume ± SEM (A) and mouse body weights ± SEM (B) of each group were monitored 2–3 times/week during therapy. CH-2-77 was given to mice 5 times a week intraperitoneally. At study endpoint, ex vivo tumor volume ± SD (C) and tumor wet weights (in grams) ± SD (D) were measured. The mean values are shown above each scatter bar graph. (E) Lung metastases ± SD of each group after manually counting nodules in H&E stained whole lung sections. The mean number of metastases is indicated above each bar. (F) Representative images of H&E stained lung images; metastases are indicated by black arrows.

Figure 6.

CH-2-77 inhibits primary tumor growth in an orthotopic MDA-MB-231 TNBC xenograft model in comparison with CA4P. Mean tumor volume ± SEM (A) and mouse body weights ± SEM (B) of each group were monitored 2–3 times/week during therapy. At study endpoint, ex vivo tumor volume ± SEM (C) and tumor wet weights (in grams) ± SEM (D) were measured. The mean values are shown above each scatter bar graph. (E) Tumor images representative of each group’s mean ex vivo tumor volume and tumor wet weight.