Chronic radiation exposure of neuroblastoma cells reduces nMYC copy number.

Neuroblastoma accounts for >15% of cancer-associated mortalities of children in the USA. Despite aggressive treatment regimens, the long-term survival for these children remains <40%. The identification of v-Myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (nMYC) gene amplification during diagnosis is associated with poor prognosis in neuroblastoma. There are limited studies examining changes in nMYC copy numbers in response to therapy and its biological effect on cancer cells. The aim of the present study was to evaluate the effect of radiation on nMYC expression and amplification status in high-risk neuroblastoma. The effect of acute (5 Gy) and chronic (25 Gy) radiation on two nMYC-amplified cell lines, SK-N-BE (2) and NB-1691, was investigated. The results demonstrate that, following chronic but not acute radiation, the two cell lines regained their proliferation potential similar to the controls. This increased proliferation was characterized by loss of nMYC mRNA and protein expression. It was also revealed that nMYC loss was accompanied by nuclear localization of c-Myc. Using fluorescent in situ hybridization and quantitative polymerase chain reaction analysis, the results of the present study demonstrated that chronic radiation causes a severe loss of nMYC gene copy number. The present study is the first to provide experimental evidence that prolonged radiation therapy affects nMYC gene copy number in high-risk neuroblastoma but does not significantly improve the prognostic outlook.

SPARC overexpression suppresses radiation-induced HSP27 and induces the collapse of mitochondrial Δψ in neuroblastoma cells.

Neuroblastoma is the cause of >15% of cancer-associated mortality in children in the USA. Despite aggressive treatment regimens, the long-term survival rate for these children remains at <40%. The current study demonstrates that secreted protein acidic and rich in cysteine (SPARC) suppresses radiation-induced expression of heat shock protein 27 (HSP27) in vivo and suppresses mitochondrial membrane potential (Δψ) in neuroblastoma cells. In the present study, the overexpression of SPARC in SK-N-BE(2) and NB1691 neuroblastoma cell lines suppresses radiation-induced G2M cell cycle arrest, proliferation, HSP27 expression (in vitro and in vivo) and induces the collapse of the mitochondrial Δψ. Gene ontology analysis demonstrated that the overexpression of SPARC combined with irradiation, induces the expression of dissimilar molecular function genes in SK-N-BE(2) and NB1691 cells, providing evidence of a dissimilar response signaling pathway. These results demonstrate that overexpression of SPARC suppresses radiation-induced HSP27 expression in neuroblastoma cells and the combination of SPARC and radiation induces the expression of protein 21, but suppresses neuroblastoma tumor density in in vivo mouse models. SPARC also induces mitochondrial Δψ collapse in SK-N-BE(2) and NB1691 neuroblastoma cells.

Matrix metalloproteinase-9 inhibition down-regulates radiation-induced nuclear factor-kappa B activity leading to apoptosis in breast tumors.

PURPOSE: Novel strategies are needed to prevent the high mortality rates of several types of cancer. These high rates stem from tumor resistance to radiation therapy, which is thought to result from the induction of matrix metalloproteinases (MMP) and plasminogen activators. In the present study, we show that the modulation of MMP-9 expression, using adenoviral-mediated transfer of the antisense MMP-9 gene (MMP-9 adenoviral construct, Ad-MMP-9), affects breast cancer sensitivity to radiation. EXPERIMENTAL DESIGN: In the present study, we used antisense Ad-MMP-9 to down-regulate the expression of MMP-9 in MDA MB 231 breast cancer cell lines in vitro before irradiation and subsequently incubated cells in hypoxic condition. In vivo studies were done with orthotopic breast tumors, and radiosensitivity was evaluated both in vitro and in vivo. RESULTS: Ad-MMP-9 infection resulted in down-regulation of radiation-induced levels of hypoxia-inducible factor 1 alpha and MMP-9 under hypoxic conditions in MDA MB 231 breast cancer cells. In addition, Ad-MMP-9, in combination with radiation, decreased levels of the transcription factors nuclear factor-kappaB and activator protein 1, both of which contribute to the radioresistance of breast tumors. Finally, the triggering of the Fas-Fas ligand apoptotic cascade, which resulted in the cleavage of PARP-1 and caspase-10, caspase-3, and caspase-7, signifies the efficiency of combined treatment of Ad-MMP-9 and radiation. Treatment with Ad-MMP-9 plus radiation completely regressed tumor growth in orthotopic breast cancer model. CONCLUSIONS: In summary, integrating gene therapy (adenovirus-mediated inhibition of MMP-9) with radiotherapy could have a synergistic effect, thereby improving the survival of patients with breast cancer.

In vitro Evaluation of Eclipta alba against Serogroups of Leptospira interrogans.

Leptospirosis is now acknowledged as the most widespread zoonoses in the world. Hundreds of cases occur in India every year accounting for considerable morbidity and sizable mortality. Several studies have delineated the epidemiology, pathology and variable clinical features of this condition. The present study comprises the importance and utilization of traditional based medicines to overcome the adverse reaction by conventional drugs and standardize the technology. The antileptospiral activity of Eclipta alba L. was well studied by both tube dilution and micro dilution techniques and the result showed better inhibitory action against various serogroups of Leptospira interrogans. L. australis, L. autumnalis and L. grippotyphosa are inhibited by both water and ethanol extract by tube dilution technique. The MIC level observed are 50 µg and 100 µg respectively. Similarly acetone extract, Icterohaemorrhagiae was responded to 200 µg/ml as MIC whereas in petroleum ether extract, no inhibition was observed. In the case of micro dilution technique, the entire inhibition rates are supported to the tube dilution technique. It showed that the micro dilution technique is the best method where we obtained the results within 30 minutes; at the same time tube dilution technique takes minimum of 7 days to provide the result.

Adenovirus-mediated small interfering RNA against matrix metalloproteinase-2 suppresses tumor growth and lung metastasis in mice.

Matrix metalloproteinases (MMP) are a group of proteinases that have normal physiologic roles degrading and remodeling the extracellular matrix. They also have multiple roles in different stages of tumor progression. Elevated levels of MMPs have been observed in many tumors; these increases have a strong association with the invasive phenotype. MMP-2 and MMP-9 are particularly involved in cancer invasion and metastasis. MMP inhibitors are currently being tested as therapeutic agents for a number of cancers in both preclinical models and in clinical trials. To date, clinical trials using this strategy have had limited efficacy. A major concern is the lack of specificity of commercially available MMP inhibitors. An adenoviral vector expressing small interfering RNA against the MMP-2 gene (Ad-MMP-2) was constructed to specifically inhibit MMP-2 expression. The effect of Ad-MMP-2 on invasion, angiogenesis, tumor growth, and metastasis of A549 lung cancer cell was evaluated. Ad-MMP-2 infection of lung cancer cells showed specific down-regulation of MMP-2 protein, activity, and transcription as determined by Western blotting, gelatin zymography, and reverse transcription-PCR. Ad-MMP-2 inhibition also mitigated lung cancer invasion and migration, and reduced tumor cell-induced angiogenesis in vitro. In an experimental metastatic lung tumor model, treatment of established tumors by Ad-MMP-2 inhibited s.c. tumor growth and formation of lung nodules in mice. Adenoviral-mediated RNA interference against MMP-2 has significant therapeutic potential for lung cancer and exerts some of this effect by inhibiting angiogenesis.

Inhibition of invasion, angiogenesis, tumor growth, and metastasis by adenovirus-mediated transfer of antisense uPAR and MMP-9 in non-small cell lung cancer cells.

Lung cancer is currently the leading cause of cancer deaths in the United States. Conventional therapeutic treatments, including surgery, chemotherapy, and radiation therapy, have achieved only limited success. The overexpression of proteases, such as urokinase-type plasminogen activator (uPA), its receptor (uPAR), and matrix metalloproteinases (MMP), is correlated with the progression of lung cancer. In the present study, we used a replication-deficient adenovirus capable of expressing antisense uPAR and antisense MMP-9 transcripts to simultaneously down-regulate uPAR and MMP-9 in H1299 cells. Ad-uPAR-MMP-9 infection of H1299 cells resulted in a dose- and time-dependent decrease of uPAR protein levels and MMP-9 activity as determined by Western blotting and gelatin zymography, respectively. Corresponding immunohistochemical analysis also showed that Ad-uPAR-MMP-9 infection inhibited uPAR and MMP-9 expression. As shown by Boyden chamber assay, Ad-uPAR-MMP-9 infection significantly decreased the invasive capacity of H1299 cells compared with mock and Ad-CMV (empty vector)-infected cells in vitro. Furthermore, Ad-uPAR-MMP-9 infection inhibited capillary-like structure formation in H1299 cells cocultured with endothelial cells in a dose-dependent manner compared with mock- and Ad-CMV-infected cells. Ad-uPAR-MMP-9 injection caused the regression of s.c. induced tumors after s.c. injection with H1299 lung cancer cells and inhibited lung metastasis in the metastatic model with A549 cells. These data suggest that Ad-uPAR-MMP-9 shows its antitumor activity against both established and early phases of lung cancer metastases by causing the destruction of the tumor vasculature. In summary, adenovirus-mediated inhibition of uPA-uPAR interaction and MMP-9 on the cell surface may be a promising anti-invasion and antimetastatic strategy for cancer gene therapy.

Interventional modalities in the treatment of complex regional pain syndrome.

Complex regional pain syndrome (CRPS) applies to a variety of conditions in which symptoms such as allodynia and hyperalgesia predominate along with hyperpathia and vasomotor/sudomotor disturbances. The incidence of CRPS in the chronic pain population varies and is difficult to determine, though it appears to affect women more than men. Treatment is multidisciplinary, and recovery of function and the reduction of pain are the main goals of treatment;this article addresses some of the interventional modalities that are used.