New flavonoid and pentacyclic triterpene from <i>Sesamum indicum</i> leaves

<div><p>Two new and one known compounds were isolated from the methanol extract of <i>Sesamum indicum</i> leaves. By means of spectroscopic methods, their structures were elucidated and identified to be 3-epibartogenic acid (<b>1</b>), kaempferol 3-<i>O</i>-[2-<i>O</i>-(<i>trans</i>-<i>p</i>-coumaroyl)<i>-3-O-α</i>-L-rhamnopyranosyl]-β-d-glucopyranoside (<b>2</b>) and epigallocatechin (<b>3</b>). Compound <b>1</b> and <b>3</b> strongly inhibited α-amylase with the IC₅₀ values of 146.7 and 303.9 μM, respectively, in comparison with acarbose (IC₅₀ 124.0 μM).</p></div

SRF cytotoxicity involves JNK kinases and proceeds via caspase-3 activation and mitochondrial membrane potential loss.

<p>(A) SRF (10 µM) activates JNK kinase but not ERK and p38. Phosphorylation status of the JNK, ERK and p38 was probed by immunoblotting using phospho-specific antibodies against the kinases. SRF selectively induces JNK phosphorylation [Panel (i)] without altering protein levels [Panel (ii)]. (B and C) Pre-treatment of cells with JNK-specific inhibitor prior to SRF (10 µM) exposure abrogates Bcl-2 and Bad phosphorylation. No phosphorylation of Bcl-2 (Panel B, Lane 4) or Bad (Panel C, Lane 3) were seen when cells were pre-treated with JNK-specific inhibitor, SP600125. Similar results were not observed with highly selective non-competitive ERK1/2 inhibitor, PD98059 (Panel B, Lane 6) or p38 inhibitor, SB203580 (Panel B, Lane 8). (D) Inhibition of JNK-kinase protects cells against SRF-induced toxicity. Cell viability was determined by MTT assay and reported as percentage control. SP600125 was able to retain viability in approximately 70% cells. Data are shown as means ± SEM. **<i>P</i><0.01 versus control. (E) Cells pre-treated with SP600125 were able to overcome SRF-induced G₂/M phase cell cycle blockage. Percentage cells in the different stages of cell cycle were determined by flow cytometric analysis. Data are shown as means ± SEM. *<i>P</i><0.05; **<i>P</i><0.01 versus control. (F) SP600125 treated cells retain cellular microtubule network. Fluorescence micrographs of cells treated with SRF in the presence or absence of SP600125. Microtubules (green) and nucleus (blue) were stained with FITC-conjugated anti-tubulin antibody and DAPI, respectively. Scale bar = 10 µM. (G) SRF induces loss of mitochondrial membrane potential as shown by flow-cytometric analysis of cells stained with JC-1. Events were counted in the green channel. SP600125 pre-treatment prevented cells from undergoing apoptosis as percentage of cells having fluorescence in the green channel decreased from 84.2% in SRF treated cells to 47.8% for cells that were pre-treated with SP600125 prior to SRF (10 µM) exposure. (H) Apoptotic death mediated by SRF proceeds through caspase-3 activation. A cleaved band corresponding to activated caspase-3 is present in SRF-treated lysates but absent from SP600125 pre-treated lysates.</p

Comparison between the effect of SRF and other microtubule binding agents on viability of drug-resistant cell types.

a<p>The KB-V1 is a mutant of the parental cell line KB-3-1 and resistant to vinblastine.</p><p>Comparison between the effect of SRF and other microtubule binding agents on viability of drug-resistant cell types.</p

The antitumoral effect of SRF on human colon adenocarcinoma HCT15 xenografts.

<p>SCID female mice were implanted with 1×10⁷ HCT15 human colon adenocarcinoma cells. The treatment started when tumor size attained a 100–200 mm³ or larger. Animals were treated intraperitoneally (i.p.) with 20 mg/kg/dose SRF in final dosing formulation or a 0.9% saline vehicle. Compound was given to tumor-bearing mice on days 1, 4, and 7 after staging, and tumor mass [(length×width²)/2] was determined once a three days for 9 days. Three tumors were observed at different time points. Error bars represent standard error of the mean (n = 3). The data were analyzed by a one-sided Student’s <i>t</i> test, and values of P<0.05 were considered to be significant. *P<0.05 versus vehicle control.</p

Cartoon representing plausible mechanism of SRF mediated toxicity in cancer cells.

<p>SRF can bypass P-gp mediated drug efflux via mechanism(s) that are currently unknown. Inside the cell, SRF binds and inhibits microtubule polymerization resulting in cell cycle arrest at the G₂/M phase. These events, in turn, activate JNK-mediated stress-response signaling cascade leading to the phosphorylation and inactivation of anti-apoptotic proteins like Bcl-2 and Bad. Consequently, there is loss of mitochondrial membrane potential and integrity, release of cytochrome-c, activation of caspase-3 and eventual cell death by apoptosis. Thus, SRF-mediated cell death proceeds via the intrinsic/mitochondrial apoptotic pathway.</p

Effect of SRF on BH-3 family members, Bcl-2 and Bad.

<p>(A) Western blot analysis of HeLa and WI-38 cell extracts treated with 10 µM SRF for 24 hr and probed with anti-Bcl-2 monoclonal antibody. A slower migrating band corresponding to phosphorylated form of Bcl-2 is present in HeLa extracts but absent from WI-38 lysates, showing that SRF induces selective phosphorylation of Bcl-2 in cancer cells. (B) SRF mediates Bcl-2 hyperphosphorylation. Drug treated HeLa lysates were resolved on 12% SDS-PAGE and immunoblotting was done using specific phospho-antibodies against T56, S70 and S87 residues of Bcl-2; all these amino acids lie in the flexible loop region of the protein. (C) Time course analysis of Bad phosphorylation induced by SRF treatment. HeLa cell lysates were analyzed by immunoblotting using anti-Bad monoclonal antibody. SRF (10 µM) treatment altered phosphorylation status of pro-apoptotic protein Bad as indicated by the appearance of a slower migrating phospho-Bad band at 24 h. The band was absent from control (DMSO treated) cells even after 24 h.</p

SRF-mediated microtubule depolymerization results in cell cycle arrest at G₂/M phase.

<p>(A) Flow cytometric analysis of the DNA content of cells treated with SRF (10 µM), nocodazole or taxol. Shown are representative one-parameter histograms of treated cells. Like taxol and nocodazole, SRF-mediated inhibition of microtubule dynamics resulted in cell cycle arrest at the G₂/M transition phase. (B) Fluorescence micrographs of mitotic cells that had been pre-treated with the indicated compounds. Compared to vehicle treated cells, SRF and other TBAs inhibited formation and segregation of the mitotic spindle. Microtubules (green) were stained with FITC-conjugated anti-tubulin antibody; nucleus (blue) was stained with DAPI. Images were acquired at 60X magnification. Scale bar = 5 µM. (C) SRF-treatment induces rapid apoptosis in proliferating cells. Following a brief 7 hr SRF exposure (10 µM), apoptotic progression was monitored by double-staining cells with Annexin V-FITC (FL-1)/PI (FL-2). During this period, percentage of annexin V⁺/PI⁻ (indicating early apoptosis) cells increased from 2.4% (in control) to 21.7%. The kinetics of this increment is similar to that seen with nocodazole and taxol. Both these compounds induced apoptosis in approximately 25.4% (nocodazole) and 33.2% (taxol) of cells within this time period. The experiments were done in triplicate (n = 3) and the data value was averaged.</p

SRF binds microtubules at the colchicine-binding site.

<p>(A) SRF competes with colchicine for binding to microtubules as was determined using competition-binding scintillation proximity assay (SPA). In comparison to control (no competitor), SRF decreased colchicine binding by 2.5-fold. Data are shown as means ± SEM. *<i>P</i><0.05, **<i>P</i><0.01 versus control. (B) SRF and vinblastine have distinct binding sites on tubulin as SRF does not compete with vinblastine. Results shown are mean (± SD) of three independent experiments. Data are shown as means ± SEM. *<i>P</i><0.05 versus control. (C–E) Cartoon representation of binding mode of colchicine (C) and SRF (D) to tubulin. The binding mode of drugs to tubulin was examined by docking studies using molecular docking program GOLD (Genetic Optimization for Ligand Docking, Cambridge Crystallographic Data Centre, UK) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110955#pone.0110955.s008" target="_blank">Methods S1</a>]. The compounds (colchicine or SRF) were docked into the colchicine-binding pocket of tubulin using the crystal structure of the tubulin-colchicine: stathmin-like domain [PDB code: 1SA0]. H-bond interactions between the indazole ring of SRF and N349 and K352 of β-chain (green) as well as the T179 and V181 of α-chain (blue) are highlighted. Panel E shows overlay of SRF and colchicine highlighting the difference in binding patterns of these molecules.</p

SRF - a novel tubulin-binding agent that depolymerizes microtubules.

<p>(A) Chemical structure of SRF. (B) Effect of SRF on microtubule polymerization <i>in vitro</i>. Purified tubulin was incubated at 37°C in the absence (DMSO) or presence of drugs like taxol (3 µM), nocodazole (10 µM), SRF and absorbance was measured at 420 nm every 1 min over a 60 min period. SRF inhibited microtubule polymerization in a concentration-dependent manner as was indicated by a decrease in absorbance with time. (C) Confocal micrographs of HeLa cells exposed to SRF, taxol and nocodazole. Cells were labeled with FITC-conjugated anti-tubulin antibody. SRF treatment completely destroys the intricate microtubule network. Scale bar = 10 µM.</p

Effect of SRF on viability of different cancer cell types.

<p>Effect of SRF on viability of different cancer cell types.</p