Neuroblastoma Cell Lines Contain Pluripotent Tumor Initiating Cells That Are Susceptible to a Targeted Oncolytic Virus

Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers.Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice.These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus

Gating of proton and water transfer in the respiratory enzyme cytochrome c oxidase

The membrane-bound enzyme cytochrome c oxidase is responsible for cell respiration in aerobic organisms and conserves free energy from O(2) reduction into an electrochemical proton gradient by coupling the redox reaction to proton-pumping across the membrane. O(2) reduction produces water at the bimetallic heme a(3)/Cu(B) active site next to a hydrophobic cavity deep within the membrane. Water molecules in this cavity have been suggested to play an important role in the proton-pumping mechanism. Here, we show by molecular dynamics simulations that the conserved arginine/heme a(3) Δ-propionate ion pair provides a gate, which exhibits reversible thermal opening that is governed by the redox state and the water molecules in the cavity. An important role of this gate in the proton-pumping mechanism is supported by site-directed mutagenesis experiments. Transport of the product water out of the enzyme must be rigidly controlled to prevent water-mediated proton leaks that could compromise the proton-pumping function. Exit of product water is observed through the same arginine/propionate gate, which provides an explanation for the observed extraordinary spatial specificity of water expulsion from the enzyme

Doxorubicin-enriched, ALDH^br mouse breast cancer stem cells are treatable to oncolytic herpes simplex virus type 1

<p>Abstract</p> <p>Background</p> <p>The primary objective of this study was to test whether oncolytic herpes simplex virus type 1 (HSV1) could eradicate chemoresistant cancer stem cells (CSCs).</p> <p>Methods</p> <p>The fluorescent aldefluor reagent-based technique was used to identify and isolate ALDH^br cells as CSCs from the 4T1 murine breast cancer cell line. The presence of ALDH^br 4T1 cells was also examined in 4T1 breast cancer transplanted in immune-competent syngeneic mice.</p> <p>Results</p> <p>Compared with ALDH^lo cells, ALDH^br cells had a markedly higher ability to form tumor spheres <it>in vitro</it> and a higher tumorigenic potential <it>in vivo</it>. ALDH^br cells also exhibited increased doxorubicin resistance <it>in vitro</it>, which correlated with a selective increase in the percentage of ALDH^br cells after doxorubicin treatment and an increased expression of P-glycoprotein (P-gp), a known chemoresistance factor. In contrast, oncolytic HSV1 was able to kill ALDH^br cells <it>in vitro</it> and even more markedly <it>in vivo</it>. Furthermore, in <it>in vivo</it> studies, systemic administration of doxorubicin followed by intratumoral injection of oncolytic HSV1 resulted in much more significant suppression of tumor growth with increased median survival period compared with each treatment given alone (p<0.05). Though more CD8⁺ T lymphocytes were induced by oncolytic HSV1, no significant specific T cell response against CSCs was detected <it>in vivo</it>.</p> <p>Conclusions</p> <p>These results suggested that the use of oncolytic HSV1 following doxorubicin treatment may help eradicate residual chemoresistant CSCs <it>in vivo</it>.</p