Half maximal inhibitory concentration (IC₅₀) values of various fractions of <i>E. longifolia</i> root methanolic extract on K-562 cell line.

<p>Half maximal inhibitory concentration (IC₅₀) values of various fractions of <i>E. longifolia</i> root methanolic extract on K-562 cell line.</p

Expression changes of apoptosis and cell cycle related genes in K562 cells.

<p>Expression levels of RNA were analyzed based on the Ct value. (A): Up-regulated genes in apoptotic pathway; (B): Down-regulated genes in apoptotic pathway; (C): Up-regulated genes in cell cycle pathway; (D): Down-regulated genes in cell cycle pathway.</p

Effect of TAF273 on the size and histological appearance of subcutaneous tumor induced by injecting the K-562 cells in nude mice.

<p>(A) Gross appearance of tumors in the control mice. (B) Gross appearance of tumors in TAF273-treated mice. (<b>C</b>): An H&E-stained tumor section (original magnification of 40×) of the control group is composed of compact sheet of aggressively proliferating viable tumor cells (VC), abundance of blood vessels (BV), and the presence of mitotic figures (MC). (<b>D</b>): The tumor section (original magnification of 40×) of TAF273 (50 mg/kg) IP- treatment revealed notable changes in tumor histology, as significant loss of compact arrangement of viable tumor cells (VC), with less number of blood vessels (BV), abundance of apoptotic cells (AC) surrounded by necrotic regions (NC) and absence of mitotic figures. (<b>E</b>): Graphical comparison of the mean apoptotic cells/microscopic field (control vs TAF273). (<b>F</b>): Graphical comparison of the mean necrotic areas (control vs TAF273) as calculated by using imageJ softwere. Values are presented as mean ± SD, (<i>n</i> = 4).</p

Colony morphology (upper panel) and Dose-response curves (lower panel).

<p>K-562 cells were treated for 48 h and 72 h with various concentrations of TAF 273, eurycomanone and imatinib (positive control). After the indicated time of treatment, cells were maintained without test materials up to 14 days. At day 14, the grown colonies were photographed and counted using light microscope. The percentages of survival fraction were then calculated. <b>A</b>): control (untreated cells); <b>B–E</b>): Imatinib (1, 0.5, 0.25 and 0.125 µg/ml, respectively); <b>F–J</b>): eurycomanone (12, 6, 3, 1.5 and 0.75 µg/ml, respectively); <b>K–O</b>): TAF273 (100, 50, 25, 12.5 and 6.25 µg/ml, respectively).</p

RNA quality and integrity.

<p>(<b>A</b>): virtual gel image showing Lane L: RNA ladder, Lane 1: no RNA sample, lanes 2–12 samples. Sample 5 and 8 were for the control and TAF273-treated cells, respectively. (<b>B</b>): electrophoregram for control sample; (<b>C</b>): electrophoregram for TAF273-treated sample.</p

Conjugation of Benzylvanillin and Benzimidazole Structure Improves DNA Binding with Enhanced Antileukemic Properties

<div><p>Benzyl-o-vanillin and benzimidazole nucleus serve as important pharmacophore in drug discovery. The benzyl vanillin (2-(benzyloxy)-3-methoxybenzaldehyde) compound shows anti-proliferative activity in HL60 leukemia cancer cells and can effect cell cycle progression at G2/M phase. Its apoptosis activity was due to disruption of mitochondrial functioning. In this study, we have studied a series of compounds consisting of benzyl vanillin and benzimidazole structures. We hypothesize that by fusing these two structures we can produce compounds that have better anticancer activity with improved specificity particularly towards the leukemia cell line. Here we explored the anticancer activity of three compounds namely 2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2MP, N-1-(2-benzyloxy-3-methoxybenzyl)-2-(2-benzyloxy-3-methoxyphenyl)-1H-benzimidazole, 2XP, and (R) and (S)-1-(2-benzyloxy-3-methoxyphenyl)-2, 2, 2-trichloroethyl benzenesulfonate, 3BS and compared their activity to 2-benzyloxy-3-methoxybenzaldehyde, (Bn1), the parent compound. 2XP and 3BS induces cell death of U937 leukemic cell line through DNA fragmentation that lead to the intrinsic caspase 9 activation. DNA binding study primarily by the equilibrium binding titration assay followed by the Viscosity study reveal the DNA binding through groove region with intrinsic binding constant 7.39 µM/bp and 6.86 µM/bp for 3BS and 2XP respectively. 2XP and 3BS showed strong DNA binding activity by the UV titration method with the computational drug modeling showed that both 2XP and 3BS failed to form any electrostatic linkages except via hydrophobic interaction through the minor groove region of the nucleic acid. The benzylvanillin alone (Bn1) has weak anticancer activity even after it was combined with the benzimidazole (2MP), but after addition of another benzylvanillin structure (2XP), stronger activity was observed. Also, the combination of benzylvanillin with benzenesulfonate (3BS) significantly improved the anticancer activity of Bn1. The present study provides a new insight of benzyl vanillin derivatives as potential anti-leukemic agent.</p> </div

The activity on caspase activation enzymes after 2XP, 3BS and betulinic acid exposure.

<p>(i) The fluorescence intensity of caspase enzymes ratio on U937 cells after exposure to 2XP, 3BS and betulinic acid (as a positive control) at different time intervals. caspase 3&7 with significant difference between the treatments and the control group at 5hrs, 7hrs and 12hrs incubation period. p<0.01, (ii) comparison caspase 8 & 9 during the maximum peak period with significant difference between the caspases, p<0.0001 and within the treatment, p=0.047, (iii) caspase 8 with significant difference between the treatments and the control (DMSO) at 3hrs and 5hrs of incubation period. p<0.01, (iv) caspase 9 with significant difference among the treatments at 3hrs, 5hrs, 7hrs and 9hrs of incubation period. p<0.01. Note: The results for 3BS and betulinic acid are not displayed for 7hrs and 9 hrs as their respective caspase 3 activation occurs at 5 hours.</p

The computational modeling studies on the four compounds.

<p>The drug modeling for, i. Bn1, ii. 2MP, iii. 2XP and iv. 3BS respectively with DNA, show the binding site of each compound with their respective DNA. </p

UV spectroscopy and viscometery analyses on the different compounds.

<p>(i) the UV Absorption at λmax for Bn1, 2MP, 2XP and 3BS during the addition of DNA, (ii) Scatchard of saturation binding of 2XP and 3BS. Analysis of absorbance data at 221 and 216 for binding of 2XP and 3BS to calf thymus DNA respectively, (iii) the saturation curve of DNA binding to 2XP and (iv) the saturation curve of DNA binding to 3BS (v) the effect of 3BS, 2XP, EtBr (positive control as DNA intercalator) and Hoechst (positive control as groove binder) on DNA viscosity.</p