Recovery of Cognitive Dysfunction via Orally Administered Redox-Polymer Nanotherapeutics in SAMP8 Mice

<div><p>Excessively generated reactive oxygen species are associated with age-related neurodegenerative diseases. We investigated whether scavenging of reactive oxygen species in the brain by orally administered redox nanoparticles, prepared by self-assembly of redox polymers possessing antioxidant nitroxide radicals, facilitates the recovery of cognition in 17-week-old senescence-accelerated prone (SAMP8) mice. The redox polymer was delivered to the brain after oral administration of redox nanoparticles via a disintegration of the nanoparticles in the stomach and absorption of the redox polymer at small intestine to the blood. After treatment for one month, levels of oxidative stress in the brain of SAMP8 mice were remarkably reduced by treatment with redox nanoparticles, compared to that observed with low-molecular-weight nitroxide radicals, resulting in the amelioration of cognitive impairment with increased numbers of surviving neurons. Additionally, treatment by redox nanoparticles did not show any detectable toxicity. These findings indicate the potential of redox polymer nanotherapeutics for treatment of the neurodegenerative diseases.</p></div

Measurement of adverse effects.

<p>(A) Effects of RNP^N on blood pressure of SAMP8 mice in tail-cuff blood pressure method. Blood pressures before administration (white bar), after single administration (black bar), one week after starting treatment (red bar), two weeks after starting treatment (green bar), three weeks after starting treatment (yellow bar), and four weeks after starting treatment (blue bar) are shown. Values are expressed as mean ± SEM (n = 10). *P < 0.05 compared with SAMP8 control mice. (B) Effects of redox polymer nanotherapeutics on AST (white bar) and ALT (black bar) levels of SAMP8 mice. Values are expressed as mean ± SEM (n = 10). ^#P < 0.05, ^##P < 0.01 compared with SAMR1 mice. *P < 0.05 compared with SAMP8 control mice.</p

The levels of MDA, protein carbonyl, 8-OHdG, NO, superoxide scavenging activity (% inhibition of superoxide anion), antioxidant enzyme activity of SOD, catalase and GPx in the hippocampus area of SAMP8 mice.

<p>The levels of MDA, protein carbonyl, 8-OHdG, NO, superoxide scavenging activity (% inhibition of superoxide anion), antioxidant enzyme activity of SOD, catalase and GPx in the hippocampus area of SAMP8 mice.</p

Concept of orally administered redox polymer nanotherapeutics for treatment of the senescence-accelerated neurodegenerative diseases.

<p>(A) Structures of redox polymers and RNP^N and (B) illustration of delivery of redox polymer to the brain after oral administration of RNP^N are shown.</p

Delivery of redox polymers to the brain after oral administration of RNP^N

<p>(A) An ESR spectrum of RNP^N before its oral administration is shown. (B-F) At 30 min after oral administration of RNP^N (300 mg/kg), ESR spectra in (B) the stomach, (C) the duodenum (D) the jejunum, (E) the ileum, and (F) the blood are shown. Red arrows indicate the ESR signal of RNP^N or redox polymers. Black arrows indicate the ESR signal of Mn²⁺ marker. (G) The biodistribution of RNP^N was determined using ¹²⁵I-labeled RNP^N. The percentage of radioactivity in the blood was determined by comparison to the injected total radioactivity. The data are expressed as the mean ± SEM values (n = 5). (H) The localization of redox polymer in the duodenum was determined after oral administration of Cy5.5-labeled RNP^N. Mice were sacrificed at 0.5 h after oral administration of 1 mL of Cy5.5-labeled RNP^N at a dose of 2 mg/mL, and the duodenum section was cut circularly. The localization of Cy5.5-labeled redox polymer in the duodenum was analyzed by fluorescent confocal microscopy (Zeiss LSM 700 under oil immersion; Scale bars = 100 μm). Lu and Se in the figure indicate lumen and serosa, respectively. Arrows indicate fluorescent signal of Cy5.5-labeled redox polymer. (I, J) Redox polymers interacted with serum proteins in the bloodstream after oral administration. (I) Interaction between redox polymers and FITC-BSA <i>in vitro</i>, determined by fluorescent quenching of FITC-BSA by nitroxide radical moieties in redox polymers (n = 1). (J) Interaction between redox polymers with serum proteins in the blood. Chromatogram of ¹²⁵I-labeled RNP^N (upper chromatogram), ¹²⁵I-labeled BSA (middle chromatogram), and the blood sample after oral administration of ¹²⁵I-labeled RNP^N (lower chromatogram). (K) The biodistribution of RNP^N in the brain via oral administration using ¹²⁵I-labeled RNP^N (white square) and ESR measurement (black circle). The data are expressed as mean ± SEM, n = 5. (L) ESR spectrum of redox polymer in the brain at 30 min after oral administration of RNP^N (500 mg/kg). Red arrows show the ESR signal of redox polymers. Black arrows show the ESR signal of Mn²⁺ marker.</p

Therapeutic effect of RNP^N on cognitive dysfunction.

<p>(A) The latency periods of saline-treated SAMR1 mice (open circle), saline-treated SAMP8 mice (open square), blank micelles-treated SAMP8 mice (closed triangle), TEMPOL-treated SAMP8 mice (closed circle), and RNP^N-treated SAMP8 mice (closed square) were measured by the Morris water-maze test. The values are expressed as mean ± SEM values (n = 10). ^#P < 0.05, ^##P < 0.01, compared with SAMR1 mice. *P < 0.05, **P < 0.01, compared with SAMP8 control mice. (B) The exploration times of saline-treated SAMR1 mice (open circle), saline-treated SAMP8 mice (open square), blank micelles-treated SAMP8 mice (closed triangle), TEMPOL-treated SAMP8 mice (closed circle), and RNP^N-treated SAMP8 mice (closed square) were measured by the object-recognition test. The values are expressed as mean ± SEM values (n = 10). ^#P < 0.05, ^##P < 0.01, compared with SAMR1 mice. *P < 0.05, **P < 0.01, compared with SAMP8 control mice.</p

Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells

<div><p>Withaferin A (WA), a major bioactive component of the Indian herb <i>Withania somnifera</i>, induces cell death (apoptosis/necrosis) in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD). WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs) in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L). Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS) in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L) suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.</p></div

Cell viability of TIG-1, LNCaP, DU-145, and PC-3 cells after WA treatment.

<p><b>(A, B)</b> Cell viability was measured at 4, 8, and 24 h after 2 μM (A) or 4 μM (B) WA treatment. NT, non-treated. Green and blue arrows indicate bars for surviving cells, whereas pink and red arrows indicate bars for cells that died under the same conditions. Yellow arrows indicate the samples used for DNA microarray analysis. Bars represent means ± SEM for three independent experiments. Purple arrows indicate significant reductions in cell viabilities (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01).</p

WA induces BAG3-mediated autophagy in PC-3 cells.

<p>A) Observation of EGFP and EGFP-LC3 signals by immunofluorescence microscopy. Bar, 10 μm. (B) Bar graphs showing the percentage of cells containing punctate EGFP-LC3; arrows show that the values gradually increased under the indicated conditions. Data are represented as the means ± SEM of three independent experiments; green arrows indicate statistically significant increases (**, <i>P</i> < 0.01). (C) Western blot analysis to detect LC-3 and GAPDH (loading control) in PC-3 under the indicated conditions. (D) Bar graphs showing cell viability (%) under the indicated conditions. (E) Western blot analysis to detect BAG3, LC-3, and GAPDH (loading control) in PC-3 cells in the presence of the indicated conditions of siBAG3 or siGL2 (negative control). (F) Bar graphs showing cell viability (%) at the indicated conditions. (D, F) Data are represented as means ± SEM of three independent experiments; red arrows indicate statistically significant reductions in cell viability (**, <i>P</i> < 0.01). (A–F) Samples were collected at 4 h after WA treatment. (G) Expression profiles of the autophagy-related proteins BAG3 and LC3 in PC-3 cells at 4, 8, and 24 h after treatment with 4 μM WA. NT: non-treated.</p

c-Fos and FLIP play a role in induction of cell death.

<p>(A, B) Western blot analysis to detect c-Fos (FosB) in TIG-1, LNCaP, DU-145, and PC-3 cells at 4, 8, and 24 h after treatment with 4 μM (A) or 2 μM (B) WA. NT, non-treated. (C) Western blot analysis to detect c-Fos, PARP, FLIP and GAPDH in PC-3 cells at 12 h after 4 μM WA treatment in the presence (+) or absence (-) of three different siRNAs (X–Z) from OriGene. (D) Viability of PC-3 cells after siFos treatment. Data are represented as means ± SEM of three independent experiments; pink arrows indicate a statistically significant reduction in cell number following siFos treatment (**, <i>P</i> < 0.01). (E) Population of apoptotic, necrotic, and live cells distinctly stained with Annexin V–EnzoGold, 7-AAD-Red, and GFP. Data are represented as means ± SEM of three independent experiments; red, blue, and green arrows indicate statistically significant changes (**, <i>P</i> < 0.01). (F) Western blot analysis of c-Fos, PARP, FLIP, and GAPDH in DU-145 at 12 h after 4 μM WA treatment in the presence (+) or absence (-) of siRNAs; siFos or siGL2 (siControl). (G) Viability of DU-145 cells after exogenous overexpression of pcDNA3-FLIP or vector alone in the presence of DMSO (solvent) or 4 μM WA.</p