Antiproliferative activity of some 1,4-dimethylcarbazoles on cells that express estrogen receptors: part I

Abstract

Several 9H-carbazole derivatives are used for various pharmacological applications. Many of these compounds demonstrated cytotoxic and anticancer activities. In this work, we have investigated the cytotoxic activity of some substituted carbazoles against cancer cell lines (MCF-7, and ISK). The derivative 2a showed the highest inhibitory activity against both cell lines.

Keywords::

• Carbazoles
• anticancer
• MCF-7 cell line
• Ishikawa cell line

Introduction

The indole nucleus is often found embedded in compounds with a wide range of biological activities¹. Carbazoles, whether synthetic or naturally occurring, are important members of indole-containing heterocycles^1,2. Several 9H-carbazoles possess various pharmacological applications and also they are used in dyes-, insecticide-, and plastic industries. In addition, several derivatives of 1,2,3,4-tetrahydro-9H-carbazole and 9-methyl-9H-carbazole have been known by their medicinal use as antitubercular, antifungal, antibacterial, β₃-adrenoceptor agonists and anticancer activities^2–4.

The precise mechanisms of their anti-neoplastic activity have not yet been explained. Many of these carbazoles are cytostatic and their activity results from alternative cytotoxic effects. It was suggested that the prevalent mechanisms of the antitumor, mutagenic and cytotoxic activities are their intercalation into DNA and inhibition of DNA-topoisomerase II activity^5,6.

Based on this, the aim of this work is to investigate the cytotoxic activity of the prepared carbazoles (Figure 1) against a cancer cell line. We have used MCF-7 cell line derived from a pleural effusion of a postmenopausal 69-year-old patient with metastatic breast cancer in 1970⁷. This cell line was grown in Dulbecco’s modified Eagle’s medium (DMEM), without phenol red, containing 10% foetal calf serum (Invitrogen, Milan, Italy) and 1 mg/mL penicillin-streptomycin. The MCF-7 cell line is the most widely used and best characterized of all the human breast cancer cell lines⁸. MCF-7 cells respond to estrogens and anti-estrogens, have differential expression of estrogen receptors (ER), progesterone receptor, and have high proliferation rates⁹. MCF-7 cells are also a perfect model to study the pathway of malignant progression as they can be subjected to appropriate endocrinologic and physiologic selective pressures for the derivation of variants with more progressed phenotypes⁸.

Figure 1.  Carbazole derivatives (1a–g).

Further in this work, some tests were performed on Ishikawa cell line (ISK) derived from an endometrial adenocarcinoma of a 39-year-old Asian woman¹⁰. This cell line was maintained in Dulbecco’s modified Eagle’s medium-F12 (DMEM/F12), without phenol red, supplemented with 10% foetal bovine serum (Invitrogen, Milan, Italy) and 1 mg/mL penicillin-streptomycin.

Ishikawa cells express estrogen¹¹, progesterone¹², androgen¹³ and aryl hydrocarbons receptors¹⁴. There is a big evidence for the hormonal responsiveness of Ishikawa cells. This evidence is based mainly on hormonal modification of proliferation, cellular functions or gene expression¹⁵. In particular, these cells are used in vitro for the elucidation of molecular mechanisms of hormone action (e.g., in drug and discovery processes), testing of potential agonistic functions of anti-estrogens or selective estrogen receptor modulators in an endometrial-derived model¹⁶, in studies of ligand independent activation of the estrogen receptor and anchorage independent tumour growth¹⁷. In addition, they are used for studies on factors controlling hormonal receptivity¹⁸ and on environmental toxicology on the function of phytoestrogens¹⁹ and endocrine-disrupting chemicals²⁰ in an endometrial model; paracrine cell/cell-interaction²¹, signalling cross-talk¹⁴ and others²². Because of their hormonal responsiveness in vitro, Ishikawa endometrial adenocarcinoma cells have also been developed as an estrogen sensitive in vivo tumour model, which is particularly suitable for the study of hormonal growth control²³.

Results and discussion

Carbazoles 1a–g were synthesized^24–27 and characterized^28–34 using earlier reported methods including ours.

In particular, the 9H-carbazoles 1a–d were prepared by the reaction of appropriate indoles with hexane-2,4-dione in the presence of p-toluenesulphonic acid. The 9H-carbazole 1e was instead prepared by reaction of 1a with ethyl chloroformate under standard conditions³⁵. Furthermore, we have used 1a as starting compound for the preparation of 1f–g. In fact, 1a was N-methylated by iodomethane and in the presence of sodium hydride to give compound 1f. The carbazole NH could be N-protected by (Boc)₂O to give 1g (Figure 1).

In order to evaluate the cytotoxic activity of prepared compounds (1a–g) against MCF-7 and ISK cell lines, an MTT assay was performed³⁶ using increasing concentrations (0.1, 1, 10 μM) of the compounds under testing. Compound 1a showed a significant inhibition at 1 and 10 μM, against both MCF-7 and ISK cell lines (Figure 2A, and B). Concerning the MCF-7 cells, a 30% inhibition was observed by the lower concentrations of 0.1 and 1 μM of 1b and 1c, respectively (Figure 2A). However, with ISK cells, compound 1b displayed a significant inhibitory effect on cell proliferation only at 10 μM concentration, while the inhibitory effect of 1c was observed at a concentration of 0.1 and 10 μM (Figure 2B). On the other hand, 1d showed an inhibitory effect against MCF-7 cells at 1 μM concentration (Figure 2A), and the same compound, however, displayed more inhibition against ISK cells at a concentration of 1 and 10 μM (Figure 2B). Compound 1e significantly reduced cell proliferation of both MCF-7 and ISK cells at a concentration of 0.1 μM and 1 μM (Figure 2A, and B).

Figure 2.  Effect of carbazole derivatives (1a–e) against MCF-7 and ISK cell proliferation. MCF-7 (A) and ISK (B) cells were treated for 96 or 24 h, respectively, after 24-h starvation, with the indicated concentrations of substances 1a, 1b, 1c, 1d and 1e. Cells proliferation was evaluated by MTT assay. Statistically significant differences are indicated. Histograms; mean of three independent experiments each performed with triplicate samples expressed as percent of basal; bars, SE (*P < 0.01 compared with basal).

Based on the above results indicating the ability of compound 1a to inhibit proliferation of both cell lines (Figure 2A, and B), structural modifications of 1a were made in order to find out whether the introduction of a substituent such as a methyl- (1f) or a Boc substituent (1g), at the 9 position of the carbazole nucleus could affect the antiproliferative activity.

Figure 3 showed that the introduction of a methyl group (an electron-donating small substituent), improved the inhibitory activity, whereas the introduction of Boc group did not show any improvement of activity. The derivative 1f showed the highest inhibitory activity against both cell lines (at low concentration of 0.1 µM) (Figure 3A, and B), the matter that encourages its use as a possible antitumor agent. However, the observation that higher concentrations (1, 10 µM) of 1f do not induce dose-response inhibitory effect on both MCF-7 and ISK cell viability suggests a possible role of 1f in blocking a survival pathway binding to receptor desensitized at high concentration. This hypothesis is currently under investigation in our laboratories.

Figure 3.  Effect of carbazole derivatives (1f–g) against MCF-7 and ISK cell proliferation. MCF-7 (A) and ISK (B) cells were treated for 96 or 24 h, respectively, after 24-h starvation, with the indicated concentrations of substances 1f and 1g. Cells proliferation was evaluated by MTT assay. Statistically significant differences are indicated. Histograms; mean of three independent experiments each performed with triplicate samples expressed as percent of basal; bars, SE (*P < 0.01 compared with basal).

Acknowledgements

We are thankful to Dr. Ewa Surmacz (Sbarro Institute for Cancer Research and Molecular Medicine, Philadelphia, PA, USA) and to Dr. D. Picard (University of Geneva, Geneva, Switzerland) for their kind offer of MCF-7 and Ishikawa cell lines, respectively.

Declaration of interest

The authors report no conflicts of interest.