The triterpenoid cucurbitacin B augments the antiproliferative activity of chemotherapy in human breast cancer

Abstract

Despite recent advances in therapy, breast cancer remains the second most common cause of death from malignancy in women. Chemotherapy plays a major role in breast cancer management, and combining chemotherapeutic agents with nonchemotherapeutic agents is of considerable clinical interest. Cucurbitacins are triterpenes compounds found in plants of the Cucurbitaceae family, reported to have anticancer and anti-inflammatory activities. Previously, we have shown antiproliferative activity of cucurbitacin B (CuB) in breast cancer, and we hypothesized that combining CuB with chemotherapeutic agents can augment their antitumor effect. Here, we show that a combination of CuB with either docetaxel (DOC) or gemcitabine (GEM) synergistically inhibited the proliferation of MDA-MB-231 breast cancer cells in vitro. This antiproliferative effect was accompanied by an increase in apoptosis rates. Furthermore, in vivo treatment of human breast cancer orthotopic xenografts in immunodeficient mice with CuB at either low (0.5 mg/kg) or high (1 mg/kg) doses in combination with either DOC (20 mg/kg) or GEM (12.5mg/kg) significantly reduced tumor volume as compared with monotherapy of each drug. Importantly, no significant toxicity was noted with low-dose CuB in combination with either DOC or GEM. In conclusion, combination of CuB at a relatively low concentration with either of the chemotherapeutic agents, DOC or GEM, shows prominent antiproliferative activity against breast cancer cells without increased toxicity. This promising combination should be examined in therapeutic trials of breast cancer.

Figure 1. Effect of CuB, DOC and GEM on proliferation of MDA-MB-231 cells in vitro

MTT-proliferation assays of MDA-MB-231 cells treated for 96 hrs with different concentrations of the following drugs: Panels (A) CuB, (B) DOC, (C) GEM, (D) CuB/DOC or (E) CuB/GEM. (F, G) Normalized isobolograms. Numbers represent different concentrations of Panel (F) CuB/DOC, Panel (G) CuB/GEM. Panel (F) #1: DOC 10⁻⁹ mol/L+ CuB 10⁻⁹ mol/L; #2: DOC 10⁻⁹ mol/L + CuB 10⁻⁸ mol/L; #4: DOC 5 × 10⁻⁹ mol/L + CuB 10⁻⁹ mol/L; #5: DOC 5 × 10⁻⁹ mol/L + CuB 10⁻⁸ mol/L; #6: DOC 10⁻⁸ mol/L + CuB 5 x 10⁻⁸ mol/L. Panel (G) #1: GEM 10⁻⁹ mol/L + CuB 10⁻⁹ mol/L; #2: GEM 10⁻⁹ mol/L + CuB 10⁻⁸ mol/L; #4: GEM 10⁻⁸ mol/L + CuB 10⁻⁹ mol/L; #7: GEM 10⁻⁷ mol/L + CuB 10⁻⁹ mol/L; #8: GEM 10⁻⁷ mol/L + CuB 10⁻⁸ mol/L; #9: GEM 10⁻⁷ mol/L + CuB 5 x 10⁻⁹ mol/L; *: p < 0.05; **: p < 0.01; ***: p< 0.001.

Figure 2. Effect of CuB, DOC and GEM on apoptosis in MDA-MB-231 cells

(A) Display of the percentage of cells in early (lower right box) and in late (upper right box) apoptosis. (B) Graphic display of the percentage of cells in early apoptosis. *: p < 0.05; **: p < 0.01.

Figure 3. CuB augmented the anti-proliferative activity of DOC and GEM in MDA-MB-231 orthotopic xenografts

MDA-MB-231 cells (10⁶) were subcutaneously injected in two mammary fat pads of each nude mouse. Mice were treated as follow: Panel (A) CuB/DOC combination: (1) diluent control (PBS, three times/week); (2) low dose CuB (0.5 mg/kg three times/week); (3) high dose CuB (1.0 mg/kg three times/week); (4) single agent DOC (20 mg/kg two times/week); (5) combination CuB (0.5 mg/kg three times/week) and DOC (20 mg/kg two times/week); (6) combination CuB (1 mg/kg three times/week) and DOC (20 mg/kg i.p. two times/week). Panel (B) CuB/GEM combination: (1) diluent control (PBS, three times/week); (2) low dose CuB (0.5 mg/kg three times/week); (3) high dose CuB (1.0 mg/kg three times/week); (4) single agent GEM (12.5 mg/kg two times/week); (5) combination CuB (0.5 mg/kg three times/week) and GEM (12.5 mg/kg i.p. two times/week); (6) combination CuB (1 mg/kg three times/week) and GEM (12.5 mg/kg two times/week). Results represent the mean tumor volume (cm³) of 10 tumors (5 mice) over 36 days of treatment.

Figure 4. Analysis of bone marrow toxicity after drug treatment measured by clonogenic assays

Bone marrow cells isolated from mice treated either with PBS (control), low dose CuB, high dose CuB, DOC, GEM, or combinations of either DOC or GEM with either low dose or high dose CuB (Comb low, Comb high, respectively; see Fig. 4 for in vivo treatment details, before harvest of bone marrow) were plated in triplicates in semi-solid methylcellulose medium. Box and whisker plots display mean colony counts +/−SD of CFU-GEMM, CFU-GM or BFU-E of three mice per treatment group. *, p < 0.05; **, p = 0.009; ***, p < 0.001.