TMPE Derived from Saffron Natural Monoterpene as Cytotoxic and Multidrug Resistance Reversing Agent in Colon Cancer Cells

Abstract

Terpenes constitute one of the largest groups of natural products. They exhibit a wide range of biological activities including antioxidant, anticancer, and drug resistance modulating properties. Saffron extract and its terpene constituents have been demonstrated to be cytotoxic against various types of cancer cells, including breast, liver, lung, pancreatic, and colorectal cancer. In the present work, we have studied anticancer properties of TMPE, a newly synthesized monoterpene derivative of β-cyclocitral-the main volatile produced by the stigmas of unripe crocuses. TMPE presented selective cytotoxic activity to doxorubicin-resistant colon cancer cells and was identified to be an effective MDR modulator in doxorubicin-resistant cancer cells. Synergy between this derivative and doxorubicin was observed. Most probably, TMPE inhibited transport activity of ABCB1 protein without affecting its expression level. Analysis of TMPE physicochemical parameters suggested it was not likely to be transported by ABCB1. Molecular modeling showed TMPE being more reactive molecule than the parental compound-β-cyclocitral. Analysis of electrostatic potential maps of both compounds prompted us to hypothesize that reduced reactivity as well as susceptibility to electrophilic attack were related to the lower general toxicity of β-cyclocitral. All of the above pointed to TMPE as an interesting candidate molecule for MDR reversal in cancer cells.

Keywords: ABCB1 transporter (P-glycoprotein); anticancer activity; colon cancer; monoterpene; multidrug resistance (MDR) reversal; saffron; β-cyclocitral.

Figure 1

Chemical structures of β -cyclocitral and ethyl 3-(2,6,6-trimethylcyclohex-1-en-1-yl) prop-2-enoate (TMPE).

Figure 2

Cytotoxic activity of β-cyclocitral (A) and TMPE (B) against LoVo, LoVo/Dx, HT29, and HT29/Dx cells. The results were shown as mean + S.D. from three independent experiments. Cell survival at the concentrations of 2.5, 5, 10, 25, and 35 µM of the studied compounds were compared to the results obtained for the control (without the compound). The statistical significance was determined using Student’s t-test (p < 0.05). Statistical significance was found only for TMPE in HT29/Dx cells (for all concentrations tested) and in LoVo/Dx cells (for concentrations equal or greater than 5 µM). Doubling times were: 36.44 ± 5.18 h for LoVo, 27.07 ± 4.53 h for LoVo/Dx, 30.02 ± 5.89 h for HT29, and 21.51 ± 3.27 h for HT29/Dx cell line.

Figure 3

Changes in cytotoxicity of doxorubicin against LoVo/Dx (A) and HT29/Dx (B) cells in the presence of 5 µM of β-cyclocitral and TMPE. The results were shown as mean ± S.D. from three independent experiments. The statistical significance between cytotoxicity observed in the presence of doxorubicin alone and doxorubicin used together with monoterpene compounds was determined using Student’s t-test (* p < 0.05).

Figure 4

The effect of β-cyclocitral (5 µM) and TMPE (5 µM) on the intracellular accumulation of doxorubicin in cultures of LoVo and LoVo/Dx (A), as well as HT29 and HT29/Dx (B) cells. The results were shown as mean ± S.D. from three independent experiments. The statistical significance was determined using Student’s t-test (* p < 0.05).

Figure 5

The influence of β-cyclocitral and TMPE on the intracellular accumulation of rhodamine 123 (Rho123) in LoVo/Dx (A) and HT29/Dx cells (B). Mean ± S.D. of three experiments are presented. The following FIR values were found to be statistically significant (p < 0.05) as compared to the control: TMPE in all concentrations in LoVo/Dx cells; TMPE in all concentrations in HT-29/Dx cells; β-cyclocitral at the highest concentration in LoVo/Dx cells; verapamil in all concentrations (apart from the lowest) in LoVo/Dx cells; verapamil in all concentrations in HT-29/Dx cells.

Figure 6

The influence of β-cyclocitral (5 µM) and TMPE (5 µM) on the ABCB1 level in LoVo/Dx and HT29/Dx cells (A). The base pair lengths of the amplified products are indicated at the left side of the gel. β-GUS was used as a reference gene. The relative level of ABCB1 expression in LoVo/Dx (B) and HT29/Dx cells (C) normalized to the control derived from non-treated cells. The results of three experiments (mean ± S.D.) are presented. The statistically significant differences from the untreated controls were determined using Student’s t-test (p < 0.05).

Figure 7

The influence of β-cyclocitral (5 µM) and TMPE (5 µM) on the ABCB1 protein expression in LoVo/Dx and HT29/Dx cells (A). Samples were analyzed by Western blot, stained with Ponceau, and probed with anti-ABCB1 and anti-β-actin antibodies. The respective molecular weights are indicated at the right side of the gel. The relative level of ABCB1 expression in LoVo/Dx (B) and HT29/Dx cells (C) normalized to the control derived from non-treated cells. The results of three experiments (mean ± S.D.) are presented. The statistically significant differences from the untreated controls were determined using Student’s t-test (p < 0.05).

Figure 8

HOMO (A,B) and LUMO (C,D) orbitals for β-cyclocitral (A,C) and TMPE (B,D) in aqueous solution. The images were obtained using SPARTAN software.

Figure 9

Electrostatic potential maps for β-cyclocitral (A) and TMPE (B) in aqueous solution. The blue color represents the positive region and the red color represents the negative region. The images were obtained using SPARTAN software.