Branched Glycerol-Based Copolymer with Ultrahigh p65 siRNA Delivery Efficiency for Enhanced Cancer Therapy

The small interfering RNA (siRNA) is emerging as a potential therapeutic for the treatment of various diseases because of the targeted gene silencing capability. The suppression of p65 expression has shown great potential in various cancer treatments. However, various substantial obstacles limit its clinical applications, including low cellular uptake, instability, and cytotoxicity of delivery vehicles. We show a highly branched and biocompatible glycerol-based copolymer (HBGC) to effectively deliver siRNA for excellent p65 gene silencing and safe lung cancer treatment in vitro and in vivo. HBGCs could be synthesized through a facile and modular one-spot Michael addition. HBGCs effectively protect siRNA in serum, enhance cellular uptake of siRNA, and show negligible cytotoxicity in various cells (A549, HeLa, HepG2, and C2C12). Additionally, the HBGC–siRNA complex potently downregulates the p65 expression in A549 cancer cells (almost the highest value of 96% in reported references) and enhances the cellular apoptosis compared to that of commercial transfection agents polyethyleneimine 25 kDa and Lipofectamine 2000. The HBGC–siRNA complex demonstrated significantly increased accumulation in the tumor sites and enhanced downregulation of p65 gene and cancer cell apoptosis. Furthermore, the tumor growth could be significantly inhibited in a subcutaneous lung tumor model with negligible adverse effects

As₂O₃ induces hyperacetylation of Hsp90 and decreases IKKα-binding to Hsp90 by immunoprecipitation and western blot assays.

<p>(A) The accumulation of acetylated Hsp90 in NCI-H929 cells were cultured with As₂O₃ (0 µM, 2.5 µM, and 5 µM) for 48 hours. (B) Hsp90-IKK interaction in NCI-H929 cells were cultured with As₂O₃ (2.5 µM) for 48 hours. (C) The IKKα protein expression in myeloma cells treated with As₂O₃ (0 µM, 2.5 µM, and 5 µM) for 48 hours.</p

As₂O₃ inhibits myeloma cells growth.

<p>As₂O₃ with different concentrations (0.1 µM∼64 µM) inhibits proliferation of (A) MM cell lines for 24 hours, 48 hours, and 72 hours and (B) MM patients' cells for 48 hours. Cell growth inhibition was measured with the CCK-8 reagent, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032215#s4" target="_blank">Methods</a>. (C) As₂O₃ induced apoptosis of NCI-H929 cells. Apoptosis rates were determined using Annexin-V/PI staining. Cells were incubated for 24 or 48 hours with 1 µM∼0 µM of As₂O₃, and the appropriate combination and analyzed by flow cytometry. Data are the mean+SD for three replicate measurements. * means statistical difference was observed between the treated group and control (P<0.05).</p

As₂O₃ inhibits TNF-α dependent p65 phosphorylation.

<p>(A) Cells were either untreated or pretreated with 2.5 µM As₂O₃ for 6 hours and then treated with 10 ng/mL TNF-α for 0, 10, 20, 30, 60, 120 minutes time points. (B) Cells were incubated with 2.5 µM, 5 µM, 7.5 µM As₂O₃ for 6 hours and then treated with 10 ng/mL TNF-α for 20 minutes. Western blot analysis was performed with anti-p65 and anti-phospho- p65-Ser 536.</p

As₂O₃ can cause a decrease in HDAC activity.

<p>Graphs in the panel are HDAC activity expressed as ODs, in the same scale as the positive and negative controls that are also HeLa cells extracts with and without trichostatin A treatment. At the bottom are values of As₂O₃ treatment concentration. * means statistical difference was observed between the treated group and control (P<0.05).</p

As₂O₃ triggers the accumulation of α-tubulin acetylation.

<p>(A) NCI-H929 cells were cultured with As₂O₃ (1 µM, 2.5 µM, 5 µM, 7.5 µM and 10 µM) for 48 hours. (B) NCI-H929 cells were cultured with As₂O₃ (2.5 µM) for the indicated time periods. (C) Primary tumor cells from MM patients were cultured with As₂O₃ (5 µM) for 12 hours.</p

Eutopic/ectopic endometrial apoptosis initiated by bilateral uterine artery occlusion: A new therapeutic mechanism for uterus-sparing surgery in adenomyosis

<div><p>The objective of the present study was to investigate differences in the expression of apoptosis-related factors in the eutopic and ectopic endometrium (EuE/EE) in women with adenomyosis before and after laparoscopic bilateral uterine artery occlusion (LUAO). Ten patients with uterine adenomyosis who received LUAO were selected as the research subjects, from whom EuE and EE tissues were obtained before and after LUAO and detected for the expression of apoptosis-related molecules in EuE and EE by PT-PCR and Western blot, and changes in the mitochondrial structure by electron microscopy. Normal endometrial stromal cells (NESC), and EuE/EE stromal cells in women with adenomyosis were cultured in a 1% O2, 5% CO2 incubator to establish a physical anoxia state in an in vitro stromal cell model. The expression of apoptosis-related molecules was observed at 0, 6, 12, 24 and 48h of hypoxic. The results showed that the expression of apoptosis-related factors in EuE and EE were increased significantly after LUAO and under hypoxic conditions in vitro, suggesting that transient ischemia and hypoxia were involved in the apoptosis of adenomysis lesions, and that uterine artery occlusion could remove adenomyosis lesions on tissue/cell level by cytoreduction, thus reaching the goal of treating adenomyosis effectively.</p></div

Western Blot showing the protein expression of apoptosis-related factors in EuE and EE before and after LUAO.

<p>(A) Difference in protein expression of apoptosis-related factors in EuE tissues before and after LUAO. (B) Difference in proteins expression of apoptosis-related factors in EE tissues before and after LUAO. (C) Corresponding grey value results in EuE Bax, caspase-3, caspase-4, caspase-8, cyt-c, Endo-G, TRADD and AIF protein expression levels were significantly increased after LUAO. (D) Corresponding grey value results in EE Bax, caspase-3, caspase-4, caspase-8, TRADD and AIF protein expression levels were significantly increased after LUAO. (E1/E2: Adenomyotic Eue abtained before and after LUAO; A1/A2: Adenomyotic EE abtained before and after LUAO. The numbers on the head of E1/E2(A1/A2) represent the 10 samples selected in this study. ^*P<0.05; ^**P<0.01; ^***P < 0.005; ns indicates P> 0.05).</p

Transmission electron microscop: changes of EuE and EE before and after LUAO (30000×).

<p>Apoptosis changes mainly include swollen mitochondria, cavitation sample change, nuclear chromatin edge, cell membrane reflex, cytoplasmic concentration, and the formation of apoptotic bodies.</p

Western Blot showing correlations between protein expression of the apoptosis-related factors.

<p>The protein expression of NESC, EuESC, EESC caspase-3 (A), Endo-G (C), caspase-8 (D), GRP78 (E) and Bax (F) was positive correlated with the duration of hypoxia in a time-dependent manner, while the protein expression of Bcl2 (B) was negatively correlated with the duration of hypoxia. (G) Western blot showing the protein expression of the apoptosis-related factors.</p