Ecdysteroid Derivatives that Reverse P-Glycoprotein-Mediated Drug Resistance

Abstract

The expression of multidrug resistance P-glycoprotein (P-gp) by cancer cells represents one of the major drawbacks to successful cancer therapy. Accordingly, the development of drugs that inhibit the activity of this transporter remains a major challenge in cancer drug discovery. In this context, several new ecdysteroid derivatives have been synthesized and evaluated as P-gp inhibitors. Two of them (compounds 9 and 14) were able to resensitize CEM^Vbl100 and LoVo^Doxo resistant cell lines to vinblastine and doxorubicin, respectively. Indeed, both compounds 9 and 14 increased the cellular accumulation of rhodamine 123 in cells expressing P-gp and stimulated basal P-glycoprotein-ATPase activity at a 1 μM concentration, demonstrating their interference with the transport of other substrates in a competitive mode. Moreover, in a medulloblastoma cell line (DAOY), compounds 9 and 14 reduced the side population representing cancer stem cells, which are characterized by a high expression of ABC drug transporters. Further, in DAOY cells, the same two compounds synergized with cisplatin and vincristine, two drugs used commonly in the therapy of medulloblastoma. Molecular docking studies on the homology-modeled structure of the human P-glycoprotein provided a rationale for the biological results, validating the binding mode within the receptor site, in accordance with lipophilicity data and observed structure-activity relationship information. Altogether, the present results endorse these derivatives as promising P-gp inhibitors, and they may serve as candidates to reverse drug resistance in cancer cells.

Figure 1

Natural ecdysteroids used as starting materials.

Scheme 1. Synthesis of Ecdysteroid 2,3–20,22 Bis-Ketals (6–9)

Reagents. General procedure A: camphosulfonic acid (0.01 mmol), 1 or 2 (0.1 mmol), appropriate anhydrous ketone (1.6 mL), 25 °C, 24–72 h.

Scheme 2. Synthesis of Ecdysteroid Esters (10–17)

Reagents. General Procedure B: 1–4 (0.1 mmol), appropriate carboxylic acid anhydride or chloride (0.5 mmol), pyridine (2.5 mL), 0 °C, 8–24 h.

Scheme 3. Synthesis of Ecdysteroid Esters (18–27)

Reagents. General Procedure C: 1–5 (0.1 mmol), N,N′-dimethylaminopyridine (0.4 mmol), triethylamine (0.4 mmol), appropriate carboxylic acid chloride (0.44 mmol), CH₂Cl₂ (2.5 mL), 0 °C, 1–4 days.

Scheme 4. Synthesis of 20-Hydroxyecdysone-2,3,22-tri(2-(1H-indol-3-yl) Acetate (28)

Reagents. 2-(1H-indol-3-yl)acetic acid (0.7 mmol), DCC (0.7 mmol), dry dioxane (3 mL), 1 h; then urea was filtered off and the filtrate added to 1 (0.1 mmol), N,N′-dimethylaminopyridine (0.05 mmol), dry dioxane (3 mL), 40 °C, 22 h.

Figure 2

Quantification presented by fold change in Rho123 fluorescence after a 2 h treatment of CEM^Vbl100 cells (panel A) and LoVo^Doxo cells (panel B), compared to untreated cells. Each compound was used at a concentration of 10 μM. Verapamil was used at concentrations of 25 and 10 μM. Data are represented as means ± SEM of three independent experiments. Statistical significance was determined using ANOVA with Newman–Keuls or Bonferroni correction. Asterisks indicate a significant difference between the new compounds and verapamil at 25 μM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

Effect of compounds 9 and 14 alone and in combination with vinblastine in CEM^Vbl100 (panels A and B) and doxorubicin in LoVo^Doxo (panels D and E) cells. Cells were treated at the indicated concentrations and fixed combination ratios, and viability was assessed by the MTT test after 48 h of incubation. Data are expressed as means ± SEM of three independent experiments. Combination indexes (CI) are calculated at the ED₅₀ and ED₇₅ for vinblastine (panel C) and doxorubicin (panel F) combination, where synergism is defined by CI < 1.

Figure 4

(A) Effects of compounds 9 and 14 on the ATPase activity of human P-gp. Each compound was tested at the concentrations of 1 and 10 μM, and verapamil at concentrations of 10 and 25 μM. The P-gp ATPase activity was expressed as fold changes, compared to untreated controls. (B) RT-PCR analysis of P-gp expression level on the CEM^Vbl100 cell line, after exposure of compounds 9 and 14 at a concentration of 10 μM for 24 h. Verapamil was used at a concentration of 25 μM. Data are expressed as means ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

(A) Flow cytometric analysis of SP cells in a medulloblastoma cell line (DAOY). Representative histograms obtained after 2 h of treatment with compounds 9 and 14 at a concentration of 10 μM. Verapamil (25 μM) was used as a positive control. (B) Quantification of SP cells under the conditions described in panel A. Data are expressed as means ± SEM. ****p < 0.0001 vs ctr. (C) MTT cell viability assay in the DAOY cell line treated with compounds 9 and 14 in combination with vincristine (VCR) or cisplatin (cisPt) for 72 h. The percentages of cell viability were normalized to untreated cells. Data are represented as the means ± SEM of at least three independent experiments. (E) The combination index (CI) calculated at the ED₅₀ for VCR with the cisplatin synergism is defined by CI < 1.

Figure 6

Binding modes for compound 9 (left, green color) and 14 (right, blue color) in the NMD (A, upper) and TMD (B, lower) domains, as obtained from docking simulations.