Figure 1.
Anti-proliferative effects of indole derivatives.
(A), Schematic diagrams of indole and its derivatives used in this study. (B), Broad-spectrum anti-proliferative effects of indole derivatives were measured in various cancer cell lines such as DBT-RG-05MG, MCF7, MDA-MB 231, MDA-MB 468, DU145, HCT116, as well as in HEK293. Cells were exposed to the compounds for 72 hr before MTT assay. The bars represent the percent (%) cell viability and standard deviation (SD) obtained from four independent experiments.
Figure 2.
(A, B and D), Figures show percent (%) chromosomal aberration and micronucleus (MN) frequency (Mean ± SEM) due to 48 hr DPDIM treatment in human lymphocytes. All the analyzed data for quantification are inserted in the respective tables. (C), Graphical representation of percentage (%) of reverse mutation on Salmonella typhimurium (TA100) in untreated (-ve control), Sodium azide treated (+ve control) and DPDIM (1 µM, 10 µM and 50 µM) treated wells in 96 well plate. (PC = positive control and *indicates p<0.001). Data are representative of three independent experiments.
Figure 3.
Evaluation of the inhibitory effect of DPDIM on EGFR pathway and induction of mitochondrial cytochrome c release.
(A), The change of Phospho-EGFR levels were examined in MCF7, MDA-MB 231 and MDA-MB 468 cells following DPDIM treatment for 24 hr by IB. (B), Alteration in normal and activated level of EGFR, HER2 and HER3 in 24 hr DPDIM treated ZR-75-1 cells were examined by IB. (C), Whole cell lysates (WCL) were prepared from 24 hr DPDIM treated cells and immunoblotted for normal and activated forms of AKT, ERK1/2 and STAT3. (D), IB analyses of Bcl-XL, Bax, Bad and Bim in MCF7, MDA-MB 231 and MDA-MB 468 cells treated with DPDIM for 24 hr. (E), Fluorescence micrographs of vehicle control (Leftmost panel) and treated (Right panels) MCF7 cells showing Cyt c release after 24 hr DPDIM treatment. All results are representative of three independent experiments.
Figure 4.
Activation of mitochondrial caspases and induction of apoptosis in DPDIM treated breast cancer cells.
(A), Activation of mitochondrial caspases-9, 7 and 3 in 24 hr DPDIM treated cells were shown by IB. (B), Analysis of PARP cleavage was done by IB in MCF7 and MDA-MB 231 cells after 24 hr treatment with DPDIM. (C), Apoptotic cell population was evaluated after 24 hr of treatment by FACS analysis using double staining with Annexin V and PI. (D), In situ TUNEL assay showing inter-nucleosomal degradation of genomic DNA in 48 hr treated MCF7 cells. Cells were stained with DAB and counterstained with methylgreen. The % TUNEL positive cells were calculated and the quantitative evaluation represented in the bar diagram with SD. *indicates P<0.0001. All results are representative of three independent experiments.
Figure 5.
Inhibition of EGF induced EGFR activation, cell viability and colony formation of MCF7 cells by DPDIM.
(A and B), Effect of DPDIM on EGFR phosphorylation and cell viability in EGF induced cells. MCF7 cells were treated with 100 ng/ml of EGF for 6 hr followed by 10 µM DPDIM for additional 24 hr. (A), Phosphorylation of EGFR in DPDIM treated and untreated cells in the presence or absence of EGF were detected by IB. Results are representative of three independent experiments. Densitometric value of each IB band is mentioned in the figure. Bar graph with SD represents the relative scanned density of phospho EGFR bands of MCF7 cells treated with or without EGF and DPDIM. (*P = 0.002, **P < 0.0001, ***P = 0.0025) (B), Cell viability of these DPDIM treated and untreated cells in the presence or absence of EGF were measured by MTT assay. The bars represent the percent (%) cell viability and standard deviation (SD) obtained from three independent experiments. *indicates P<0.0001 (C), 100 ng/ml of EGF induced or uninduced MCF7 cells treated with either 1 µM or 10 µM DPDIM were plated at a density of 5000 cells per 35 mm dish. EGF/DPDIM or both were fed every alternative day with media exchange. After incubation of 14 days plates were examined under a microscope and representative photographs of the colonies were taken. (D), The average diameters of the colonies were computed and represented by bar graph (n = 3, ***P = 0.0101, **P = 0.0019, *P = 0.003) with SD. (E), Colonies with the diameter of ∼100 µm were counted from three individual 20X microscopic fields. Average number of colonies for each treated and untreated plates were plotted as bar graphs with their representative SD (***P = 0.0078, **P = 0.0048, *P < 0.0001).
Figure 6.
Binding analysis of DPDIM with EGFR.
(A), Ribbon representation of human EGFR kinase domain docked with DPDIM into its ATP binding site. Arrow shows the binding of DPDIM to the receptor. (B), Two-dimensional representation of DPDIM-EGFR binding interactions. Green solid lines represents hydrophobic interactions, π-cation interaction is shown in green dotted line and the hydrogen bonding with black dotted line. Residues were numbered according to the PDB ID 1M17. (C) and (D) display the thermodynamic analysis of statistical ensemble of the ligand/receptor complex. (C), Energy spectrum distribution of system states (observed conformations) as determined by molecular docking computation and (D), concordant cluster distribution of the ensemble structures over the energy axis. Conformations within 2 Å root mean square deviation (irrespective of their binding energy) were clustered together. The graphs were plotted with OriginPro 8.
Figure 7.
Reduction of breast tumor growth by DPDIM in animal model.
Six tumor bearing Sprague Dawley rats were taken for each of the DPDIM treated and untreated group and six normal rats were also taken as control. (A), Inhibition of DMBA-induced breast tumor growth after DPDIM (5 mg/kg) treatment for every alternative days upto 21 days in rats was shown. (n = 6 in each group). Data is represented as mean ± SEM and *indicates P<0.001. (B), Tumor growth curve of rats after oral administration of DPDIM for 21 days was represented in graph. Growth pattern of tumors for another 21 days after discontinuation of DPDIM treatment was also represented in graph. Representation of growth pattern of untreated tumors for 42 days is there in the graph. For each time point a group of 6 rats were taken. Bars represent standard deviation of the volume of tumors at each time point. (C), Time-course of plasma concentration over 48 hr following oral administration of 5 mg/kg DPDIM to the tumor bearing rats was shown in the figure. Mean compound concentration in rat (n = 6) plasma at each time point (at 0.5, 1, 2, 3, 4, 5, 8, 16, 24 and 48 hrs) was studied by HPLC analysis on a Shimadzu Model SPD-M10Avp equipped with LC-10ATvp HPLC pump, Hamamatsu Deuterium Lamp type L6585 photodiode array detector. Main parameters studied for this compound in rat was given in the inserted table. Cmax = Maximum plasma concentration of a drug after oral administration; tmax = Time to reach Cmax; AUC = Area under the curve; Kel = elimination rate constant and T1/2 = Biological half life.
Figure 8.
In vivo determination of DPDIM induced EGFR pathway regulation directed to apoptosis.
(A), Expression and activation of EGFR, AKT, STAT3, ERK1/2, Bcl-XL, Bax and Caspases-3, 7 and 9 in tumor tissue lysates isolated from untreated and treated groups were checked by IB. (B and C), Tissue sections were prepared from tumors excised at day 21. (B), IHC analysis of phospho-EGFR, Bcl-XL and cleaved caspase-3 was shown in figure. Percentage of positive cells per microscopic field of 5 different fields for each antibody were calculated and represented as a bar diagram with SD. * indicates P<0.0001. (C), H&E staining were done for the histological analysis. TUNEL assay of normal breast, untreated and treated tumor tissue sections. All images were captured under bright field microscope with 20X magnification.