Novel Resveratrol and 5-Fluorouracil Coencapsulated in PEGylated Nanoliposomes Improve Chemotherapeutic Efficacy of Combination against Head and Neck Squamous Cell Carcinoma

Increasing consumption of tobacco and alcohol has led to a steady increase in the incidence of head and neck cancers in Asia. The drawbacks associated with the existing chemotherapeutic and surgical interventions have necessitated the development of a safer alternative for therapy of head and neck cancers. In this study we have explored the synergistic therapeutic potential of a phytochemical and chemotherapeutic agent using PEGylated liposomes as a delivery vehicle. Resveratrol and 5-fluorouracil were successfully coencapsulated in a single PEGylated nanoliposome. The thermal analysis and the nuclear magnetic resonance results revealed that resveratrol localized near the glycerol backbone of the liposomal membrane while 5-fluorouracil localized closer to the phosphate moiety, which influenced the release kinetics of both drugs. The nanoformulation was tested in vitro on a head and neck cancer cell line NT8e and was found to exhibit a GI50 similar to that of free 5-fluorouracil. Further, gene expression studies showed that the combination of resveratrol and 5-fluorouracil exhibited different effects on different genes that may influence the net antagonistic effect. The coencapsulation of resveratrol and 5-fluorouracil in a liposomal nanocarrier improved the cytotoxicity in comparison with the free drug combination when tested in vitro

Sirtuin assay of purified Hos2 preparations using resveratrol: UPDATE 1

<p>Fluorimetric Sirtuin1 like deacetylase assay with rHos2 protein using Fluor de Lys®−Sirt1 substrate in presence of Sirtuin1 activator Reserveratrol. This experiment is carried out in presence of HDAC inhibitor Trichostatin to inhibit any residual Histone deacetylase activity. Updated from: http://dx.doi.org/10.6084/m9.figshare.841658.</p

Deacetylase activities of rHos2 and dose response curves of rHos2 inhibition by standard HDAC inhibitors: UPDATE 1

<p>Dose response curve of rHos2 enzyme inhibition by MS275: Fluorimetric deacetylase assay with rHos2 protein using Boc-Lys(ac)-AMC substrate in presence of different concentrations of MS-275.</p> <p>Dose response curve of rHos2 enzyme inhibition by SAHA: Fluorimetric deacetylase assay with rHos2 protein using Boc-Lys(ac)-AMC substrate in presence of different concentrations of suberoylanilide hydroxamic acid (SAHA).</p> <p>Dose response curve of rHos2 enzyme inhibition by TSA: Fluorimetric deacetylase assay with rHos2 protein using Boc-Lys(ac)-AMC substrate in presence of different concentrations of trichostatin.</p> <p>rHos2 conc_Enzyme activity: Fluorimetric deacetylase assay with rHos2 protein using Boc-Lys(ac)-AMC substrate. Updated from: http://dx.doi.org/10.6084/m9.figshare.841655</p

Tubulin deacetylation and rHos2 protein blots

<p>Western blot analysis of rHos2 protein polyclonal anti-sera: Western blot analysis of rHos2 protein using polyclonal antisera raised from mice.</p> <p>Tubulin Deacetylation Blot data_F1000: Western blot analysis for tubulin Deacetyation by rHos2 protein.</p

A high-throughput cidality screen for Mycobacterium tuberculosis.

Exposure to Mycobacterium tuberculosis (Mtb) aerosols is a major threat to tuberculosis (TB) researchers, even in bio-safety level-3 (BSL-3) facilities. Automation and high-throughput screens (HTS) in BSL3 facilities are essential for minimizing manual aerosol-generating interventions and facilitating TB research. In the present study, we report the development and validation of a high-throughput, 24-well 'spot-assay' for selecting bactericidal compounds against Mtb. The bactericidal screen concept was first validated in the fast-growing surrogate Mycobacterium smegmatis (Msm) and subsequently confirmed in Mtb using the following reference anti-tubercular drugs: rifampicin, isoniazid, ofloxacin and ethambutol (RIOE, acting on different targets). The potential use of the spot-assay to select bactericidal compounds from a large library was confirmed by screening on Mtb, with parallel plating by the conventional gold standard method (correlation, r2 = 0.808). An automated spot-assay further enabled an MBC90 determination on resistant and sensitive Mtb clinical isolates. The implementation of the spot-assay in kinetic screens to enumerate residual Mtb after either genetic silencing (anti-sense RNA, AS-RNA) or chemical inhibition corroborated its ability to detect cidality. This relatively simple, economical and quantitative HTS considerably minimized the bio-hazard risk and enabled the selection of novel vulnerable Mtb targets and mycobactericidal compounds. Thus, spot-assays have great potential to impact the TB drug discovery process

Spot-assay correlation in Msm.

<p>To check suitability of the assay. <b>a</b>. <b>MBC correlation</b> showing inter-assay variability (n = 3) in the cfu-based assay in a 25-μl volume in Msm using RIOE. <b>b</b>. <b>Correlation of spot <i>vs</i>. cfu in Msm</b> for RIOE. MBC values were deduced from this finding (<b>a</b>.) and were compared with the data obtained by the conventional method. The spot-assay MBC of four RIOE reference drugs on Msm was compared with the standard cfu-based plating assay. Cfu-based MBC: Minimum concentration that yielded a ≥2 log₁₀ reduction from the starting cfu. Spot-based MBC: Minimum concentration that resulted in no growth (NG) or countable colonies (upto 30 colonies). The data for all four drugs correlated well for both methods (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117577#pone.0117577.g005" target="_blank">Fig. 5b</a>). <b>c</b>. <b>Correlation of spot <i>vs</i>. cfu of Mtb</b> for RIOE. This method was subsequently replicated in Mtb for all four RIOE reference drugs.</p

Unravelling the Secrets of Mycobacterial Cidality through the Lens of Antisense

<div><p>One of the major impediments in anti-tubercular drug discovery is the lack of a robust grammar that governs the in-vitro to the in-vivo translation of efficacy. <i>Mycobacterium tuberculosis</i> (Mtb) is capable of growing both extracellular as well as intracellular; encountering various hostile conditions like acidic milieu, free radicals, starvation, oxygen deprivation, and immune effector mechanisms. Unique survival strategies of Mtb have prompted researchers to develop in-vitro equivalents to simulate in-vivo physiologies and exploited to find efficacious inhibitors against various phenotypes. Conventionally, the inhibitors are screened on Mtb under the conditions that are unrelated to the in-vivo disease environments. The present study was aimed to (1). Investigate cidality of Mtb targets using a non-chemical inhibitor antisense-RNA (AS-RNA) under in-vivo simulated in-vitro conditions.(2). Confirm the cidality of the targets under in-vivo in experimental tuberculosis. (3). Correlate in-vitro <i>vs</i>. in-vivo cidality data to identify the in-vitro condition that best predicts in-vivo cidality potential of the targets. Using cidality as a metric for efficacy, and AS-RNA as a target-specific inhibitor, we delineated the cidality potential of five target genes under six different physiological conditions (replicating, hypoxia, low pH, nutrient starvation, nitrogen depletion, and nitric oxide).In-vitro cidality confirmed in experimental tuberculosis in BALB/c mice using the AS-RNA allowed us to identify cidal targets in the rank order of <i>rpoB>aroK>ppk>rpoC>ilvB</i>. <i>RpoB</i> was used as the cidality control. In-vitro and in-vivo studies feature <i>aroK</i> (encoding shikimate kinase) as an in-vivo mycobactericidal target suitable for anti-TB drug discovery. In-vitro to in-vivo cidality correlations suggested the low pH (R = 0.9856) in-vitro model as best predictor of in-vivo cidality; however, similar correlation studies in pathologically relevant (Kramnik) mice are warranted. In the acute infection phase for the high fidelity translation, the compound efficacy may also be evaluated in the low pH, in addition to the standard replication condition.</p></div

Spot-assay correlation in Msm.

<p>To check suitability of the assay. <b>a</b>. <b>MBC correlation</b> showing inter-assay variability (n = 3) in the cfu-based assay in a 25-μl volume in Msm using RIOE. <b>b</b>. <b>Correlation of spot <i>vs</i>. cfu in Msm</b> for RIOE. MBC values were deduced from this finding (<b>a</b>.) and were compared with the data obtained by the conventional method. The spot-assay MBC of four RIOE reference drugs on Msm was compared with the standard cfu-based plating assay. Cfu-based MBC: Minimum concentration that yielded a ≥2 log₁₀ reduction from the starting cfu. Spot-based MBC: Minimum concentration that resulted in no growth (NG) or countable colonies (upto 30 colonies). The data for all four drugs correlated well for both methods (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117577#pone.0117577.g005" target="_blank">Fig. 5b</a>). <b>c</b>. <b>Correlation of spot <i>vs</i>. cfu of Mtb</b> for RIOE. This method was subsequently replicated in Mtb for all four RIOE reference drugs.</p

Spot-assay correlation in Mtb and the Manhattan analysis.

<p>The spot-assay was replicated in Mtb for RIOE. <b>a. An MBC correlation between the spot-assay and the conventional method</b>: A strong positive correlation (r² = 0.808). <b>b & c: The Manhattan analysis</b> of isoniazid-treated reference controls exhibited variation within the 2-fold in the spot-assay (<b>b</b>) and conventional MBC methods (<b>c</b>).</p