Indole and 2,4-Thiazolidinedione conjugates as potential anticancer modulators

Abstract

Background: Thiazolidinediones (TZDs), also called glitazones, are five-membered carbon ring molecules commonly used for the management of insulin resistance and type 2 diabetes. Recently, many prospective studies have also documented the impact of these compounds as anti-proliferative agents, though several negative side effects such as hepatotoxicity, water retention and cardiac issues have been reported. In this work, we synthesized twenty-six new TZD analogues where the thiazolidinone moiety is directly connected to an N-heterocyclic ring in order to lower their toxic effects.

Methods: By adopting a widely applicable synthetic method, twenty-six TZD derivatives were synthesized and tested for their antiproliferative activity in MTT and Wound healing assays with PC3 (prostate cancer) and MCF-7 (breast cancer) cells.

Results: Three compounds, out of twenty-six, significantly decreased cellular viability and migration, and these effects were even more pronounced when compared with rosiglitazone, a well-known member of the TZD class of antidiabetic agents. As revealed by Western blot analysis, part of this antiproliferative effect was supported by apoptosis studies evaluating BCL-xL and C-PARP protein expression.

Conclusion: Our data highlight the promising potential of these TZD derivatives as anti-proliferative agents for the treatment of prostate and breast cancer.

Keywords: Apoptosis; BCL-xL; Cancer; Cell proliferation; Cellular viability; Thiazolidinediones; Wound healing.

Figure 1. Synthesis strategy of TZD compounds.

Figure 2. Structures of synthetized TZD analogues.

Figure 3. Effects of TZD compounds (AC1–AC26) on cell viability.

MTT assays were performed in MCF7 (A) and PC3 (B) cells as reported in the Materials and Methods section. Optical density (OD) was measured at 540 nm, after 24, 48 and 72 h drug treatment. Results are the mean ± S.E. of triplicates from three independent experiments. *p < 0.05, **p < 0.01 relative to untreated control cells (C = 1), which received dosing vehicle alone (0.001% DMSO). The selected AC18, AC20 and AC22 TZD analogues, which were the most active in the inhibition of cell viability in MTT assays, are shown.

Figure 4. Comparison between Rosiglitazone, AC18, AC20 and AC22 on cell viability.

MTT assays were performed in MCF7 (A) and PC3 (B) cells, either untreated or treated as indicated. OD was measured at 540 nm, after 24, 48 and 72 h drug exposure. Results are the mean ± S.E. of triplicates from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 relative to either untreated control cells (C = 1), or cells treated with the non-effective AC1 compound. Representative Western blots of BCL-xL and C-PARP from cell extracts of untreated and treated MCF-7 and PC3 cells are shown in the autoradiograms, together with densitometric results of BCL-xL and C-PARP proteins over b-Actin. All protein samples were processed at the same time, under the same experimental conditions, PVDF membranes simultaneously exposed to ECL detection reagent, and immunocomplexes visualized by enhanced chemiluminescence in the dark for 1 min. Autoradiograms were generated by positioning the membranes in the same X-ray cassette and thus exposed to the same film, in the same exposure conditions. Rosi, rosiglitazone.

Figure 5. Inhibition of cell migration.

Wound healing assays were carried out in MCF7 (A) and PC3 (B) cells, using 200 µL pipette tips to scratch confluent cells on the base of a 12-well plate. Wound healing (% wound closure) was measured and analyzed with the NIH ImageJ software in both cell types, after 4, 8 and 12 h incubation with the compounds. Results are the mean ± SE of triplicates from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 relative to untreated (control) cells.

Figure 6. In silico Induced Fit Docking of TZD compounds into the PPARγ binding pocket.

Rosiglitazone (A) is shown as light green carbon sticks, while AC18 (B) AC20 (C) and AC22 (D) are shown as green, white and cyan ball-and-sticks, respectively. The residues located within 4 Å from the bound ligand are displayed (gray sticks) and labeled. Hydrogen bonds, bad and ugly contacts between the ligands and the enzyme residues or water molecules are depicted with yellow, orange and red dashed arrows, respectively.