br Since previous studies have established that the indanone
Since previous studies have established that the indanone ring ex-erts anti-cancer activity (Ganellin, 1967; Klaus, 1983; Vilums et al., 2015; Yao et al., 2003), here we investigate the anti-cancer eﬀects of a series of indanone-based thiazolyl hydrazones on diﬀerent human cancer cell lines. Moreover, in this study we explored the mechanism of action of most active derivative which caused the inhibition of colon cancer cells’ proliferation, produced Fulvestrant arrest and induced apoptosis. The eﬀects of this derivative on tubulin polymerization, production of reactive oxygen species (ROS), and glutathione depletion were also determined.
2. Materials and methods
2.1. Chemicals and equipment
All the thiazolyl hydrazone derivatives were synthesized at the University of Karachi, Pakistan (Supplementary Fig. 1). Regorafenib was obtained from Bayer HealthCare Pharmaceuticals Inc. (Whippany, NJ) and irinotecan hydrochloride from (Alfa Aesar, MA). A stock so-lution (10 mM) of all the compounds in DMSO was prepared and a series of dilutions were made. Fig. 1A shows the chemical structure of ITH-6. Dulbecco’s modified Eagle’s Medium (DMEM, IX), fetal bovine serum (FBS), Phosphate Buﬀer Saline (PBS), 10,000 IU/ml penicillin and 10,000 μg/ml streptomycin, and trypsin 0.25% were purchased from Hyclone (Waltham, MA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-di-phenyl-tetrazolium bromide) (MTT), Dimethyl Sulfoxide (DMSO), and other chemicals were obtained from Sigma-Aldrich Chemical Co (St. Louis, MO). Propidium Iodide (PI)/RNase staining buﬀer was pur-chased from BD biosciences (SanJose, CA) and the apoptosis kit was
purchased from Biotium (Hayward, CA). 5-(and-6)-chloromethyl-20,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) was purchased from Molecular probes™ (Eugene, OR). GSH kit from Abcam (Cambridge, MA) and HTS-Tubulin Polymerization Assay Biochem Kit from Cytoskeleton (Denver, CO).
2.2. Cell lines and cell culture
HEK293 (human embryonic kidney cell line), 3T3 (mouse fibro-blast) and human colon cancer cell lines (COLO 205, HCT-15, SW620, KM 12, HT-29) were used in this study. SW620 cell line was obtained from Dr. Susan E. Bates (Columbia University, New York) and HEK293 from Suresh Ambudkar (NIH, MD). All other cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA). The cell lines were cultured at 37 °C, 5% CO2 with DMEM containing 10% FBS and 1% penicillin/streptomycin.
2.3. Cell proliferation assay
The anti-cancer eﬀects of ITH-6, regorafenib and irinotecan were determined by a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) calorimetric assay (Śliwka et al., 2016). Cells were cultured, counted and seeded onto 96 well plates with a cell density of 6 × 103 cells per well. Following 24 h incubation, the cells were treated with diﬀerent drugs (ranging from a concentration of 0–30 μM). After 68 h, 20 μl of 4 mg/ml MTT, was added to each well and the plates were further incubated 37 °C for 4 h. Subsequently, the MTT was removed from all wells and 100 μl of DMSO was added to dissolve the formazan crystals formed by the reduction of MTT in the mitochondria of the viable cells. The optical density (OD) was measured at 570 nm by an Opsys microplate reader (Dynex technologies, VA).
2.4. Cell cycle analysis
Based on the cytotoxic eﬀects of ITH-6, the cell cycle analysis was carried out on colon cancer cell lines HT-29, COLO 205, and KM 12. The cells were treated with ITH-6 at three diﬀerent concentrations (0.3, 1 and 3 μM) for 24 h and the cell cycle analysis was performed as
Fig. 1. Chemical structure of N-Indan-1-yli-dene-N'-(4-Biphenyl-4-yl-thiazol-2-yl)-hy-drazine (ITH-6) and cytotoxicity of ITH-6, Regorafenib and Irinotecan in HT-29, COLO 205, and KM 12 cell lines. The chemical structure of ITH-6 was drawn using Chem Draw (A). Survival fraction (%) (B) was mea-sured after treatment with ITH-6 for 72 h in HT-29 (orange), COLO 205 (blue), and KM 12 (grey) cell lines. Points with error bars re-present the mean ± SD for independent de-terminations in triplicate. The figures are re-presentative of three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
S. Narayanan, et al.
Table 1 The eﬀect of ITH-6 on normal cell lines.
Compounds Code HEK293 3T3
μM-Micromole. The cytotoxic eﬀects of the test compounds on HEK293 (human embryonic kidney cells) and 3T3 (mouse fibroblasts).
Values in tables are representative of at least three independent experiments performed in triplicates. IC50: concentration that inhibits cell survival by 50% (mean ± SD).