Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function-2

. An aliquot of cells was used to examine efficiency of C19ORF5 depletion (, upper panel) and NORE1A expression level (, lower panel) while other cells growing on coverslip were fixed, processed for immunofluorescence and imaged as described in Methods (). In , each panel shows NORE1A, microtubules (stained with α-tubulin), centrosomes (stained with pericentrin) and a superimposed image. Arrows indicate centrosomes. Bar, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function"</p><p>http://www.biomedcentral.com/1756-0500/1/13</p><p>BMC Research Notes 2008;1():13-13.</p><p>Published online 15 May 2008</p><p>PMCID:PMC2518271.</p><p></p

Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function-3

Day cells were infected with retrovirus encoding GFP-NORE1A. Two days later, cells were sorted using GFP and Cy3 markers, allowed to recover overnight, lysed and equal amounts of cell extract were probed for expression of MAP1B protein (, upper panel) and GFP-NORE1A expression level (, lower panel). Lysates of GFP and Cy3- positive cells were examined for the ability of GFP-NORE1A to interact with microtubules as described in Figure 1 . : A549 cells expressing GFP-NORE1A were transfected with anti-MAP1B siRNA pool or control siRNA. Cells were fixed, processed for immunofluorescence, and imaged as described in Methods. Each panel shows NORE1A, microtubules (stained with α-tubulin), centrosomes (stained with pericentrin) and a superimposed image. Arrows indicate centrosomes. Bar, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function"</p><p>http://www.biomedcentral.com/1756-0500/1/13</p><p>BMC Research Notes 2008;1():13-13.</p><p>Published online 15 May 2008</p><p>PMCID:PMC2518271.</p><p></p

Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function-4

En infected with retrovirus expressing GFP or GFP-NORE1A effector domain (aa 191–363). Two days later, cells were fixed, processed for flow cytometry and analyzed as described in Methods. The percentage of GFP-positive cells in each phase of the cell cycle +/- mean is shown.<p><b>Copyright information:</b></p><p>Taken from "Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function"</p><p>http://www.biomedcentral.com/1756-0500/1/13</p><p>BMC Research Notes 2008;1():13-13.</p><p>Published online 15 May 2008</p><p>PMCID:PMC2518271.</p><p></p

Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function-1

S were probed for C19ORF5 (upper panel) or used to examine the ability of NORE1A to bind to microtubules using the microtubule cosedimentation assay. : HEK293 cell extract was immunodepleted of the C19ORF5 protein by incubation with 4G1 antibody followed by Protein A/G plus agarose, or mock-depleted. Equal amounts of C19ORF5-depleted and mock-depleted extracts were probed for C19ORF5 (upper panel). The ability of purified FLAG-NORE1A to interact with microtubules after preincubation with C19ORF5-immunodepleted extract (lanes 1–2), mock-immunodepleted extract (lanes 3–4), or purified FLAG-C19ORF5 (lanes 5–6) was examined as described in the Methods. . C19ORF5 protein was depleted by RNA interference in A549 cells expressing GFP-tagged NORE1A. Equal amounts of cell extracts were probed for C19ORF5 (upper panel) or used to examine the ability of NORE1A to bind to microtubules as described in Figure 1 (two middle panels). Lower panel, the ability of GFP moiety, expressed alone in A549 cells, to bind to microtubules was examined.<p><b>Copyright information:</b></p><p>Taken from "Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function"</p><p>http://www.biomedcentral.com/1756-0500/1/13</p><p>BMC Research Notes 2008;1():13-13.</p><p>Published online 15 May 2008</p><p>PMCID:PMC2518271.</p><p></p

Targeting Breast Tumors with pH (Low) Insertion Peptides

Extracellular acidity is associated with tumor progression. Elevated glycolysis and acidosis promote the appearance of aggressive malignant cells with enhanced multidrug resistance. Thus, targeting of tumor acidity can open new avenues in diagnosis and treatment of aggressive tumors and targeting metastatic cancers cells within a tumor. pH (low) insertion peptides (pHLIPs) belong to the class of pH-sensitive agents capable of delivering imaging and/or therapeutic agents to cancer cells within tumors. Here, we investigated targeting of highly metastatic 4T1 mammary tumors and spontaneous breast tumors in FVB/N-Tg (MMTV-PyMT)­634Mul transgenic mice with three fluorescently labeled pHLIP variants including well-characterized WT-pHLIP and, recently introduced, Var3- and Var7-pHLIPs. The Var3- and Var7-pHLIPs constructs have faster blood clearance than the parent WT-pHLIP. All pHLIPs demonstrated excellent targeting of the above breast tumor models with tumor accumulation increasing over 4 h postinjection. Staining of nonmalignant stromal tissues in transgenic mice was minimal. The pHLIPs distribution in tumors showed colocalization with 2-deoxyglucose and the hypoxia marker, Pimonidazole. The highest degree of colocalization of fluorescent pHLIPs was shown to be with lactate dehydrogenase A, which is related to lactate production and acidification of tumors. In sum, the pHLIP-based targeting of breast cancer presents an opportunity to monitor metabolic changes, and to selectively deliver imaging and therapeutic agents to tumors

pH-sensitive pHLIP^® coated niosomes

<p>Nanomedicine is becoming very popular over conventional methods due to the ability to tune physico-chemical properties of nano vectors, which are used for encapsulation of therapeutic and diagnostic agents. However, the success of nanomedicine primarily relies on how specifically and efficiently nanocarriers can target pathological sites to minimize undesirable side effects and enhance therapeutic efficacy. Here, we introduce a novel class of targeted nano drug delivery system, which can be used as an effective nano-theranostic for cancer. We formulated pH-sensitive niosomes (80–90 nm in diameter) using nonionic surfactants Span20 (43–45 mol%), cholesterol (50 mol%) and 5 mol% of pH (Low) insertion peptide (pHLIP) conjugated with DSPE lipids (DSPE-pHLIP) or hydrophobic fluorescent dye, pyrene, (Pyr-pHLIP). In coating of niosomes, pHLIP was used as an acidity sensitive targeting moiety. We have demonstrated that pHLIP coated niosomes sense the extracellular acidity of cancerous cells. Intravenous injection of fluorescently labeled (R18) pHLIP-coated niosomes into mice bearing tumors showed significant accumulation in tumors with minimal targeting of kidney, liver and muscles. Tumor-targeting niosomes coated with pHLIP exhibited 2–3 times higher tumor uptake compared to the non-targeted niosomes coated with PEG polymer. Long circulation time and uniform bio-distribution throughout the entire tumor make pHLIP-coated niosomes to be an attractive novel delivery system.</p

pHLIP-FIRE, a Cell Insertion-Triggered Fluorescent Probe for Imaging Tumors Demonstrates Targeted Cargo Delivery <i>In Vivo</i>

We have developed an improved tool for imaging acidic tumors by reporting the insertion of a transmembrane helix: the pHLIP-<u>F</u>luorescence <u>I</u>nsertion <u>RE</u>porter (pHLIP-FIRE). In acidic tissues, such as tumors, peptides in the pHLIP family insert as α-helices across cell membranes. The cell-inserting end of the pHLIP-FIRE peptide has a fluorophore–fluorophore or fluorophore–quencher pair. A pair member is released by disulfide cleavage after insertion into the reducing environment inside a cell, resulting in dequenching of the probe. Thus, the fluorescence of the pHLIP-FIRE probe is enhanced upon cell-insertion in the targeted tissues but is suppressed elsewhere due to quenching. Targeting studies in mice bearing breast tumors show strong signaling by pHLIP-FIRE, with a contrast index of ∼17, demonstrating (i) direct imaging of pHLIP insertion and (ii) cargo translocation <i>in vivo</i>. Imaging and targeted cargo delivery should each have clinical applications