Effect of substituents at the C3´, C3´N, C10 and C2-meta-benzoate positions of taxane derivatives on their activity against resistant cancer cells

Abstract

We tested the effect of substituents at the (1) C3´, C3´N, (2) C10, and (3) C2-meta-benzoate positions of taxane derivatives on their activity against sensitive versus counterpart paclitaxel-resistant breast (MCF-7) and ovarian (SK-OV-3) cancer cells. We found that (1) non-aromatic groups at both C3´ and C3´N positions, when compared with phenyl groups at the same positions of a taxane derivative, significantly reduced the resistance of ABCB1 expressing MCF-7/PacR and SK-OV-3/PacR cancer cells. This is, at least in the case of the SB-T-1216 series, accompanied by an ineffective decrease of intracellular levels in MCF-7/PacR cells. The low binding affinity of SB-T-1216 in the ABCB1 binding cavity can elucidate these effects. (2) Cyclopropanecarbonyl group at the C10 position, when compared with the H atom, seems to increase the potency and capability of the derivative in overcoming paclitaxel resistance in both models. (3) Derivatives with fluorine and methyl substituents at the C2-meta-benzoate position were variously potent against sensitive and resistant cancer cells. All C2 derivatives were less capable of overcoming acquired resistance to paclitaxel in vitro than non-substituted analogs. Notably, fluorine derivatives SB-T-121205 and 121,206 were more potent against sensitive and resistant SK-OV-3 cells, and derivatives SB-T-121405 and 121,406 were more potent against sensitive and resistant MCF-7 cells. (4) The various structure-activity relationships of SB-T derivatives observed in two cell line models known to express ABCB1 favor their complex interaction not based solely on ABCB1.

Keywords: C10 taxane derivatives; C2 taxane derivatives; C3´ and C3´N taxane derivatives; Resistant breast cancer cells; Resistant ovarian cancer cells.

Fig. 1.

(A) Chemical structure of tested taxanes. R₁ and R₂ represent substituents at the C3´and C3´N positions, R₃ represents substituents at the C10 position, and R₄ represents substituents at the C2 position. (B) Substituents at the C3, C3´N, C10 and C2 (R₁, R₂, R₃ and R₄) positions are described.

Fig. 2.

Effect of paclitaxel on the growth and survival of original paclitaxel-sensitive SK-OV-3 cells (SEN) and paclitaxel-resistant SK-OV-3/PacR cells (RES) after 96 hours of cultivation. Each point represents the mean ± SEM (N=2, four technical replicates). The number of viable cells in a paclitaxel-free medium is not shown but was used as the upper limit for curve modeling.

Fig. 3.

Expression of ABCB1 membrane transporter in original paclitaxel-sensitive SK-OV-3 cells and paclitaxel-resistant SK-OV-3/PacR cells. (A) ABCB1 mRNA expression level is plotted as delta-delta threshold cycle (ΔΔCt) value (N=3, three technical replicates) with a 95% confidence interval. The dashed line represents ABCB1 expression in SK-OV-3 cells. Statistical significance was tested by a one-sample t-test. ***P<0.001. (B) Western blot analysis of ABCB1 transporter. β-actin served as a loading control.

Fig. 4.

Effect of taxane and taxane derivatives on the growth and survival of paclitaxel-sensitive MCF-7 and paclitaxel-resistant MCF-7/PacR breast cancer cells. The number of living cells was determined after 96 h of incubation (see “Materials and methods”). Each point represents the relative mean of two separate experiments (performed in quadruplicate) ± SEM.

Fig. 5.

Effect of taxane and taxane derivatives on the growth and survival of paclitaxel-sensitive SK-OV-3 and paclitaxel-resistant SK-OV-3/PacR ovarian cancer cells. The number of living cells was determined after 96 h of incubation (see “Materials and methods”). Each point represents the relative mean of two separate experiments (performed as quadruplicate) ± SEM.

Fig. 6.

The intracellular level of the SB-T-1216 series and paclitaxel in paclitaxel-resistant MCF-7/PacR cells relative to taxane-sensitive (control) MCF-7 cells. Each column represents the mean of peak area (normalized to total protein) in MCF-7/PacR cells relative to control paclitaxel-sensitive MCF-7 cells (N = 3, ± SEM). Statistical analysis was performed using one sample T-test. **p < 0.01, *p < 0.05 mean statististically significant result.

Fig. 7.

Comparison of paclitaxel position (green sticks) in the ABCB1 (pink cartoon) cryo-em structure 6qex with the predicted most stable binding modes of paclitaxel, SB-T-0035 and SB-T-1212, SB-T-1216 and SB-T-1214 series (colored sticks). The ABCB1 residues within 5.0 Å from paclitaxel are shown as pink lines. The predicted most stable binding modes tend to occupy a cavity near the Leu65 residue.