Sirolimus attenuates disease progression in an orthologous mouse model of human autosomal dominant polycystic kidney disease

In autosomal dominant polycystic kidney disease (ADPKD), abnormal proliferation of tubular cells drives cyst development and growth. Sirolimus, an inhibitor of the protein kinase mammalian target of rapamycin (mTOR) and a potent anti-proliferative agent, decreases cyst growth in several genetically distinct rodent models of polycystic kidney disease (PKD). We determined here the effect of sirolimus on renal cyst growth in Pkd2WS25/− mice; an ortholog of human ADPKD involving mutation of the Pkd2 gene. In Pkd2WS25/− mice treated with sirolimus, both the two kidney/total body weight (2K/TBW) ratio and the cyst volume density (CVD) were significantly decreased by over half compared with untreated mice suffering with PKD. However, there was no effect on the increased blood urea nitrogen (BUN) levels as an index of kidney function. There are two distinct complexes containing mTOR depending on its binding partners: mTORC1 and mTORC2. Western blot analysis of whole kidney lysates and immunohistochemistry of the cysts found that phospho-S6 ribosomal protein, a marker of mTORC1 activity, was increased in Pkd2WS25/− mice and its phosphorylation was decreased by sirolimus treatment. Phospho-Akt at serine 473, a marker associated with mTORC2 activity, was not different between Pkd2WS25/− mice and normal littermate controls. Hence, our study found that inhibition of mTORC1 by sirolimus correlated with decreased renal cyst growth in this model of human ADPKD but had no effect on the decline in renal function

Interleukin-1<img src='/image/spc_char/beta.gif' border=0>-induced iNOS expression in human lung carcinoma A549 cells: Involvement of STAT and MAPK pathways

840-847For understanding of signaling molecules important in lung cancer growth and progression, IL-1 effect was analyzed on iNOS expression and key signaling molecules in human lung carcinoma A549 cells and established the role of specific signaling molecules by using specific chemical inhibitors. IL-1 exposure (10 ng/ml) induced strong iNOS expression in serum starved A549 cells. Detailed molecular analyses showed that IL-1 increased expression of phosphorylated STAT1 (Tyr701 and Ser727) and STAT3 (Tyr705 and Ser727) both in total cell lysates and nuclear lysates. Further, IL-1b exposure strongly activated MAPKs (ERK1/2, JNK1/2 and p38) and Akt as well as increased nuclear levels of NF-κB and HIF-1 in A549 cells. Use of specific chemical inhibitors for JAK1 kinase (piceatannol), JAK2 kinase (AG-490), MEK1/2 (PD98059) and JNK1/2 (SP600125) revealed that IL-1-induced iNOS expression involved signaling pathways in addition to JAK-STAT and ERK1/2-JNK1/2 activation. Overall, these results suggested that instead of specific pharmacological inhibitors, use of chemopreventive agents with broad spectrum efficacy to inhibit IL-1-induced signaling cascades and iNOS expression would be a better strategy towards lung cancer prevention and/or treatment

Stage-Specific Effect of Inositol Hexaphosphate on Cancer Stem Cell Pool during Growth and Progression of Prostate Tumorigenesis in TRAMP Model

Herein, we assessed the stage-specific efficacy of inositol hexaphosphate (IP6, phytic acid), a bioactive food component, on prostate cancer (PCa) growth and progression in a transgenic mouse model of prostate cancer (TRAMP). Starting at 4, 12, 20, and 30 weeks of age, male TRAMP mice were fed either regular drinking water or 2% IP6 in water for ~8&ndash;15 weeks. Pathological assessments at study endpoint indicated that tumor grade is arrested at earlier stages by IP6 treatment; IP6 also prevented progression to more advanced forms of the disease (~55&ndash;70% decrease in moderately and poorly differentiated adenocarcinoma incidence was observed in advanced stage TRAMP cohorts). Next, we determined whether the protective effects of IP6 are mediated via its effect on the expansion of the cancer stem cells (CSCs) pool; results indicated that the anti-PCa effects of IP6 are associated with its potential to eradicate the PCa CSC pool in TRAMP prostate tumors. Furthermore, in vitro assays corroborated the above findings as IP6 decreased the % of floating PC-3 prostaspheres (self-renewal of CSCs) by ~90%. Together, these findings suggest the multifaceted chemopreventive-translational potential of IP6 intervention in suppressing the growth and progression of PCa and controlling this malignancy at an early stage

Hypoxia-inducible factor-1α (HIF-1α) and autophagy in polycystic kidney disease (PKD)

Cyst expansion in polycystic kidney disease (PKD) results in localized hypoxia in the kidney that may activate hypoxia-inducible factor-1α (HIF-1α). HIF-1α and autophagy, a form of programmed cell repair, are induced by hypoxia. The purposes were to determine HIF-1α expression and autophagy in rat and mouse models of PKD. HIF-1α was detected by electrochemiluminescence. Autophagy was visualized by electron microscopy (EM). LC3 and beclin-1, markers of autophagy, were detected by immunoblotting. Eight-week-old male heterozygous (Cy/+) and 4-wk-old homozygous (Cy/Cy) Han:SPRD rats, 4-wk-old cpk mice, and 112-day-old Pkd2WS25/− mice with a mutation in the Pkd2 gene were studied. HIF-1α was significantly increased in massive Cy/Cy and cpk kidneys and not smaller Cy/+ and Pkd2WS25/− kidneys. On EM, features of autophagy were seen in wild-type (+/+), Cy/+, and cpk kidneys: autophagosomes, mitophagy, and autolysosomes. Specifically, autophagosomes were found on EM in the tubular cells lining the cysts in cpk mice. The increase in LC3-II, a marker of autophagosome production and beclin, a regulator of autophagy, in Cy/Cy and cpk kidneys, followed the same pattern of increase as HIF-1α. To determine the role of HIF-1α in cyst formation and/or growth, Cy/+ rats, Cy/Cy rats, and cpk mice were treated with the HIF-1α inhibitor 2-methoxyestradiol (2ME2). 2ME2 had no significant effect on kidney volume or cyst volume density. In summary, HIF-1α is highly expressed in the late stages of PKD and is associated with an increase in LC3-II and beclin-1. The first demonstration of autophagosomes in PKD kidneys is reported. Inhibition of HIF-1α did not have a therapeutic effect

Effect of colistin on caspase-3/7 activity.

<p>Caspase-3/7 activity was analyzed using a Promega Caspase Glo 3/7 kit (described in the Materials and Methods). Caspase-3/7 activity was not increased at 3 h at each colistin concentration. After 6h, colistin (more than 50 μg/ml) increased caspase-3/7 activity. NAC (500 μM) antioxidant attenuated colistin-induced caspase-3/7 activity. The data represented the mean ± SEM (n = 6 for each group). *P < 0.05 vs. control cells at each time point.</p

The effect of Colistin on HK-2 cell viability.

<p>The cell toxicity of colistin was found to be dose dependent. The data represented the mean ± SEM from 8 replicates in each group. *P < 0.01 at each time point compared to control.</p

PCR Array RT²Profiler results showing genes that were significantly upregulated (The dose of colistin was 25 μg/ml).

<p>PCR Array RT²Profiler results showing genes that were significantly upregulated (The dose of colistin was 25 μg/ml).</p